Cation-based bioassay using human metabotropic glutamate receptors

ABSTRACT

In accordance with the present invention, there are provided nucleic acids encoding human metabotropic glutamate receptor subtypes and the proteins encoded thereby. In a particular embodiment, the invention nucleic acids encode mGluR1, mGluR2, mGluR3 and mGluR5 subtypes of human metabotropic glutamate receptors. In addition to being useful for the production of metabotropic glutamate receptor subtypes, these nucleic acids are also useful as probes, thus enabling those skilled in the art, without undue experimentation, to identify and isolate related human receptor subunits. In addition to disclosing novel metabotropic glutamate receptor subtypes, the present invention also comprises methods for using such receptor subtypes to identify and characterize compounds which affect the function of such receptors, e.g., agonists, antagonists, and modulators of glutamate receptor function.

RELATED APPLICATIONS

This application is a 371 of International application PCT/US94/06273,filed June 3, 1994, which is in turn a continuation-in-part applicationof U.S. Application Ser. No. 08/072,574, filed June 4, 1993, now U.S.Pat. No. 5,521,297, the entire contents of which are hereby incorporatedby reference.

The present invention relates to nucleic acids and receptor proteinsencoded thereby. Invention nucleic acids encode novel human metabotropicglutamate receptor subtypes. The invention also relates to methods formaking such receptor subtypes and for using the receptor proteins inassays designed to identify and characterize compounds which affect thefunction of such receptors, e.g., agonists, antagonists, and allostericmodulators of human metabotropic glutamate receptors.

BACKGROUND OF THE INVENTION

The amino acid L-glutamate is a major excitatory neurotransmitter in themammalian central nervous system. Anatomical, biochemical andelectrophysiological analyses suggest that glutamatergic systems areinvolved in a broad array of neuronal processes, including fastexcitatory synaptic transmission, regulation of neurotransmitterreleases, long-term potentiation, learning and memory, developmentalsynaptic plasticity, hypoxic-ischemic damage and neuronal cell death,epileptiform seizures, as well as the pathogenesis of severalneurodegenerative disorders. See generally, Monaghan et al., Ann. Rev.Pharmacol. Toxicol. 29:365-402 (1980). This extensive repertoire offunctions, especially those related to learning, neurotoxicity andneuropathology, has stimulated recent attempts to describe and definethe mechanisms through which glutamate exerts its effects.

Currently, glutamate receptor classification schemes are based onpharmacological criteria. Glutamate has been observed to mediate itseffects through receptors that have been categorized into two maingroups: ionotropic and metabotropic. Ionotropic glutamate receptorscontain integral cation-specific, ligand-gated ion channels, whereasmetabotropic glutamate receptors are G-protein-coupled receptors thattransduce extracellular signals via activation of intracellular secondmessenger systems. Ionotropic receptors are further divided into atleast two categories based on the pharmacological and functionalproperties of the receptors. The two main types of ionotropic receptorsare NMDA (N-methyl-D-aspartate) receptors and kainate/AMPA(α-amino-3-hydroxy-5-methyl-4-isoxazole propionate, formerly called thequisqualic acid or QUIS receptor), receptors. While the metabotropicreceptors bind to some of the same ligands that bind to ionotropicglutamate receptors, the metabotropic receptors alter synapticphysiology via GTP-binding proteins and second messengers such as cyclicAMP, cyclic GMP, diacylglycerol, inositol 1,4,5-triphosphate and calcium[see, for example, Gundersen et al., Proc. R. Soc. London Ser. 221:127(1984); Sladeczek et al., Nature 317:717 (1985); Nicoletti et al., J.Neurosci. 6:1905 (1986); Sugiyama et al., Nature 325:531 (1987)].

The electrophysiological and pharmacological properties of metabotropicglutamate receptors have been studied using animal tissues and celllines as a source of receptors, as well as non-human recombinantreceptors. The value of such studies for application to the developmentof human therapeutics has been limited by the availability of onlynon-human receptors. Moreover, it is only recently that thecharacteristics and structure of metabotropic glutamate receptors havebeen investigated at the molecular level. Such investigation has,however, only been carried out in non-human species. Because of thepotential physiological and pathological significance of metabotropicglutamate receptors, it is imperative (particularly for drug screeningassays) to have available human sequences (i.e., DNA, RNA, proteins)which encode representative members of the various glutamate receptorclasses. The availability of such human sequences will also enable theinvestigation of receptor distribution in humans, the correlation ofspecific receptor modification with the occurrence of various diseasestates, etc.

BRIEF DESCRIPTION OF THE INVENTION

The present invention discloses novel nucleic acids encoding humanmetabotropic glutamate receptor protein subtypes and the proteinsencoded thereby. In a particular embodiment the novel nucleic acidsencode full-length mGluR1, mGluR2, mGluR3 and mGluR5 subtypes of humanmetabotropic glutamate receptors, or portions thereof. In addition tobeing useful for the production of metabotropic glutamate receptorsubtype proteins, these nucleic acids are also useful as probes, thusenabling those skilled in the art, without undue experimentation, toidentify and isolate nucleic acids encoding related receptor subtypes.

In addition to disclosing novel metabotropic glutamate receptor proteinsubtypes, the present invention also comprises methods for using suchreceptor subtypes to identify and characterize compounds which affectthe function of such receptors, e.g., agonists, antagonists, andmodulators of glutamate receptor function. The invention also comprisesmethods for determining whether unknown protein(s) are functional asmetabotropic glutamate receptor subtypes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents a restriction map of CMV promoter-based vector pCMV-T7-2and

FIG. 2 presents a restriction map of CMV promoter-based vectorpCMV-T7-3.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided isolatednucleic acids encoding human metabotropic glutamate receptor subtypes.In one aspect of the present invention, nucleic acids encoding humanmetabotropic glutamate receptors of the mGluR1 subtype are provided. Inanother aspect, nucleic acids encoding at least a portion ofmetabotropic glutamate receptors of the mGluR2 subtype are provided. Inyet another aspect, nucleic acids encoding metabotropic glutamatereceptors of the mGluR3 subtype are provided. In a further aspect,nucleic acids encoding metabotropic glutamate receptors of the mGluR5subtype are provided. In a still further aspect, eukaryotic cellscontaining such nucleic acids, and eukaryotic cells expressing suchnucleic acids are provided.

Also provided are protein(s) encoded by the above-described nucleicacids, as well as antibodies generated against the protein(s). In otheraspects of the present invention, there are provided nucleic acid probescomprising metabotropic glutamate receptor subtype-selective portions ofthe above-described nucleic acids.

As employed herein, the phrase "human metabotropic glutamate receptorsubtypes" refers to isolated and/or purified proteins which participatein the G-protein-coupled response of cells to glutamatergic ligands.Such receptor subtypes are individually encoded by distinct genes whichdo not encode other metabotropic glutamate receptor subtypes (i.e., eachsubtype is encoded by a unique gene). Such receptor subtypes aretypically characterized by having seven putative transmembrane domains,preceded by a large putative extracellular amino-terminal domain andfollowed by a large putative intracellular carboxy-terminal domain.Metabotropic glutamate receptors share essentially no amino acidsequence homology with other G-protein-coupled receptors that are notmetabotropic glutamate receptors.

Regarding the inter-relationship between each of the metabotropicglutamate receptor subtypes, the amino acid sequences of mGluR1 receptorsubtypes are generally less than about 70% identical to the amino acidsequences of other human metabotropic glutamate receptor subtypes, withidentities less than about 45% typically observed. The amino acidsequences of mGluR2 receptor subtypes are generally less than 60%identical to the amino acid sequences of other human metabotropicglutamate receptor subtypes, with identities of less than 45% typicallyobserved. The amino acid sequences of mGluR3 receptor subtypes aregenerally less than 60% identical to the amino acid sequences of otherhuman metabotropic glutamate receptor subtypes, with identities of lessthan 45% typically observed. The amino acid sequences of mGluR5 receptorsubtypes are generally less than 70% identical to the amino acidsequences of other human metabotropic glutamate receptor subtypes, withidentities of less than 45% typically observed.

Also included within the above definition are variants thereof encodedby mRNA generated by alternative splicing of a primary transcript, aswell as fragments thereof which retain one or more of the abovephysiological and/or physical properties.

Use of the terms "isolated" or "purified" in the present specificationand claims as a modifier of DNA, RNA, polypeptides or proteins meansthat the DNA, RNA, polypeptides or proteins so designated have beenproduced in such form by the hand of man, and thus are separated fromtheir native in vivo cellular environment. As a result of this humanintervention, the recombinant DNAs, RNAs, polypeptides and proteins ofthe invention are useful in ways that the DNAs, RNAs, polypeptides orproteins as they naturally occur are not, such as identification ofselective drugs or compounds.

The term "functional", when used herein as a modifier of receptorprotein(s) of the present invention, means that binding of glutamatergicligands (such as ACPD or ACPD-like ligands, QUIS, AP4, and the like) tosaid receptor protein(s) modifies the receptor interaction withG-proteins, which in turn affects the levels of intracellular secondmessengers, leading to a variety of physiological effects. Statedanother way, "functional" means that a response is generated as aconsequence of agonist activation of receptor protein(s).

As used herein, a splice variant refers to variant metabotropicglutamate receptor subtype-encoding nucleic acid(s) produced bydifferential processing of primary transcript(s) of genomic DNA,resulting in the production of more than one type of mRNA. cDNA derivedfrom differentially processed primary transcript will encodemetabotropic glutamate receptor subtypes that have regions of completeamino acid identity and regions having different amino acid sequences.Thus, the same genomic sequence can lead to the production of multiple,related mRNAs and proteins. Both the resulting mRNAs and proteins arereferred to herein as "splice variants".

Accordingly, also contemplated within the scope of the present inventionare nucleic acids that encode metabotropic glutamate receptor subtypesas defined above, but that by virtue of degeneracy of the genetic codedo not necessarily hybridize to the disclosed nucleic acids underspecified hybridization conditions. Such subtypes also form functionalreceptors, as assessed by methods described herein or known to those ofskill in the art. Typically, unless a metabotropic glutamate receptorsubtype is encoded by RNA that arises from alternative splicing (i.e., asplice variant), metabotropic glutamate receptor subtype-encodingnucleic acids and the metabotropic glutamate receptor protein encodedthereby share substantial sequence homology with at least one of themetabotropic glutamate receptor subtype nucleic acids (and proteinsencoded thereby) described herein. It is understood that DNA or RNAencoding a splice variant may share less than 90% overall sequencehomology with the DNA or RNA provided herein, but include regions ofnearly 100% homology to a DNA fragment described herein, and encode anopen reading frame that includes start and stop codons and encodes afunctional metabotropic glutamate receptor subtype.

Exemplary DNA sequences encoding human mGluR1 subtypes are representedby nucleotides which encode substantially the same amino acid sequenceas set forth in Sequence ID No. 2. Presently preferred sequences encodethe amino acid sequence set forth in Sequence ID No. 2.

Exemplary DNA can alternatively be characterized as those nucleotidesequences which encode an human mGluR1 subtype and hybridize underhigh-stringency conditions to substantially the entire sequence ofSequence ID No. 1, or substantial portions thereof (i.e., typically atleast 25-30 contiguous nucleotides thereof).

Stringency of hybridization is used herein to refer to conditions underwhich polynucleic acid hybrids are stable. As known to those of skill inthe art, the stability of hybrids is reflected in the meltingtemperature (T_(m)) of the hybrids. T_(m) can be approximated by theformula:

    81.5° C.-16.6(log.sub.10 [Na.sup.+ ])+0.41(% G+C)-600/l,

where l is the length of the hybrids in nucleotides. T_(m) decreasesapproximately 1-1.5° C. with every 1% decrease in sequence homology. Ingeneral, the stability of a hybrid is a function of sodium ionconcentration and temperature. Typically, the hybridization reaction isperformed under conditions of lower stringency, followed by washes ofvarying, but higher, stringency. Reference to hybridization stringencyrelates to such washing conditions. Thus, as used herein:

(1) HIGH STRINGENCY conditions, with respect to fragment hybridization,refer to conditions that permit hybridization of only those nucleic acidsequences that form stable hybrids in 0.018M NaCl at 65° C. (i.e., if ahybrid is not stable in 0.018M NaCl at 65° C., it will not be stableunder high stringency conditions, as contemplated herein). Highstringency conditions can be provided, for example, by hybridization in50% formamide, 5× Denhart's solution, 5× SSPE, 0.2% SDS at 42° C.,followed by washing in 0.1× SSPE, and 0.1% SDS at 65° C.;

(2) MODERATE STRINGENCY conditions, with respect to fragmenthybridization, refer to conditions equivalent to hybridization in 50%formamide, 5× Denhart's solution, 5× SSPE, 0.2% SDS at 42° C., followedby washing in 0.2× SSPE, 0.2% SDS, at 65° C.; and

(3) LOW STRINGENCY conditions, with respect to fragment hybridization,refer to conditions equivalent to hybridization in 10% formamide, 5×Denhart's solution, 6× SSPE, 0.2% SDS at 42° C., followed by washing in1× SSPE, 0.2% SDS, at 50° C.

(4) HIGH STRINGENCY conditions, with respect to oligonucleotide (i.e.,synthetic DNA≦about 30 nucleotides in length) hybridization, refer toconditions equivalent to hybridization in 10% formamide, 5× Denhart'ssolution, 6× SSPE, 0.2% SDS at 42° C. followed by washing in 1× SSPE,and 0.2% SDS at 50° C.

It is understood that these conditions may be duplicated using a varietyof buffers and temperatures and that they are not necessarily precise.

Denhart's solution and SSPE (see, e.g., Sambrook, Fritsch, and Maniatis,in: Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, 1989) are well known to those of skill in the art asare other suitable hybridization buffers. For example, SSPE is pH 7.4phosphate-buffered 0.18M NaCl. SSPE can be prepared, for example, as a20× stock solution by dissolving 175.3 g of NaCl, 27.6 g of NaH₂ PO₄ and7.4 g EDTA in 800 ml of water, adjusting the pH to 7.4, and then addingwater to 1 liter. Denhart's solution (see, Denhart (1966) Biochem.Biophys. Res. Commun. 23:641) can be prepared, for example, as a 50×stock solution by mixing 5 g Ficoll (Type 400, Pharmacia LKBBiotechnology, INC., Piscataway, N.J.), 5 g of polyvinylpyrrolidone, 5 gbovine serum albumin (Fraction V; Sigma, St. Louis, Mo.) water to 500 mland filtering to remove particulate matter.

Especially preferred sequences encoding human mGluR1 subtypes are thosewhich have substantially the same nucleotide sequence as the codingsequences in Sequence ID No. 1; with polynucleic acid having the samesequence as the coding sequence in Sequence ID No. 1 being mostpreferred.

As used herein, the phrase "substantial sequence homology" refers tonucleotide sequences which share at least about 90% identity, and aminoacid sequences which typically share more than 95% amino acid identity.It is recognized, however, that proteins (and DNA or mRNA encoding suchproteins) containing less than the above-described level of homologyarising as splice variants or that are modified by conservative aminoacid substitutions (or substitution of degenerate codons) arecontemplated to be within the scope of the present invention.

The phrase "substantially the same" is used herein in reference to thenucleotide sequence of DNA, the ribonucleotide sequence of RNA, or theamino acid sequence of protein, that have slight and non-consequentialsequence variations from the actual sequences disclosed herein. Speciesthat are substantially the same are considered to be equivalent to thedisclosed sequences and as such are within the scope of the appendedclaims. In this regard, "slight and non-consequential sequencevariations" mean that sequences that are substantially the same as theDNA, RNA, or proteins disclosed and claimed herein are functionallyequivalent to the human-derived sequences disclosed and claimed herein.Functionally equivalent sequences will function in substantially thesame manner to produce substantially the same compositions as thehuman-derived nucleic acid and amino acid compositions disclosed andclaimed herein. In particular, functionally equivalent DNAs encodehuman-derived proteins that are the same as those disclosed herein orthat have conservative amino acid variations, such as substitution of anon-polar residue for another non-polar residue or a charged residue fora similarly charged residue. These changes include those recognized bythose of skill in the art as those that do not substantially alter thetertiary structure of the protein.

Exemplary DNA sequences encoding a portion of an human mGluR2 receptorsubtype are represented by nucleotides which encode substantially thesame amino acid sequence as set forth in Sequence ID No. 4 (optionallyincluding some or all of the 343 nucleotides of 3' untranslated sequenceset forth in sequence ID No. 13), or substantially the same amino acidsequence as that encoded by the human mGluR2-encoding portion of cloneMETAB40, deposited with the ATCC on May 4, 1993, under accession number75465.

The deposited clone has been deposited on May 4, 1993, at the AmericanType Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md.,U.S.A. 20852, under the terms of the Budapest Treaty on theInternational Recognition of Deposits of Microorganisms for Purposes ofPatent Procedure and the Regulations promulgated under this Treaty.Samples of the deposited material are and will be available toindustrial property offices and other persons legally entitled toreceive them under the terms of the Treaty and Regulations and otherwisein compliance with the patent laws and regulations of the United Statesof America and all other nations or international organizations in whichthis application, or an application claiming priority of thisapplication, is filed or in which any patent granted on any suchapplication is granted. In particular, upon issuance of a U.S. patentbased on this or any application claiming priority to or incorporatingthis application by reference thereto, all restriction upon availabilityof the deposited material will be irrevocably removed.

Presently preferred polynucleic acid sequences that encode a portion ofan human mGluR2 receptor subtype are those that encode the same aminoacid sequence as Sequence ID No. 4, or the same amino acid sequence asthat encoded by the human mGluR2-encoding portion of clone METAB40,deposited with the ATCC on May 4, 1993, under accession number 75465.

Exemplary DNAs can alternatively be characterized as those nucleotidesequences which encode a human mGluR2 receptor subtype and hybridizeunder high-stringency conditions to Sequence ID No. 3, or substantialportions thereof (i.e., typically at least 25-30 contiguous nucleotidesthereof), or the human mGluR2-encoding portion of clone METAB40 (ATCCaccession No. 75465), or substantial portions thereof. Especiallypreferred sequence encoding a portion of an human mGluR2 receptorsubtype is represented by polynucleic acid which has the same nucleotidesequence as the coding sequence set forth in Sequence ID No. 3, or thenucleotide sequence of the coding sequence in the human mGluR2-encodingportion of clone METAB40.

Exemplary DNA sequences encoding human mGluR3 receptor subtypes arerepresented by nucleotides which encode substantially the same aminoacid sequence as set forth in Sequence ID No. 6. Presently preferredpolynucleic acid sequences are those that encode the same sequence asSequence ID No. 6.

Exemplary DNAs can alternatively be characterized as those nucleotidesequences which encode a human mGluR3 receptor subtype and hybridizeunder high-stringency conditions to substantially the entire sequence ofSequence ID No. 5, or substantial portions thereof (i.e., typically atleast 25-30 contiguous nucleotides thereof). Especially preferredsequences encoding human mGluR3 subtypes are those which havesubstantially the same nucleotide sequence as the coding sequences inSequence ID No. 5, with the polynucleic acid having the same nucleotidesequence as the coding sequence set forth in Sequence ID No. 5 being thepresently most preferred.

Exemplary DNA sequences encoding human mGluR5 receptor subtypes orportions thereof are represented by nucleotides which encodesubstantially the same amino acid sequence as set forth in Sequence IDNos. 8, 10 or 12. Presently preferred polynucleic acid sequences arethose that encode the same sequence as Sequence ID Nos. 8, 10 or 12.

Exemplary DNAs can alternatively be characterized as those nucleotidesequences which encode a human mGluR5 receptor subtype and hybridizeunder high stringency conditions to substantially the entire sequence ofSequence ID Nos. 7, 9 or 11, or substantial portions thereof (i.e.,typically at least 25-30 contiguous nucleotides thereof). Especiallypreferred sequences encoding human mGluR5 subtypes are those which havesubstantially the same nucleotide sequence as the coding sequences setforth in Sequence ID Nos. 7, 9 or 11; with polynucleic acids having thesame sequence as the coding sequence set forth in Sequence ID Nos. 7, 9or 11 being the presently most preferred.

DNA encoding human metabotropic glutamate receptor subtypes may beisolated by screening suitable human cDNA or human genomic librariesunder suitable hybridization conditions with DNA disclosed herein(including nucleotides derived from any of SEQ ID Nos. 1, 3, 5, 7, 9 or11). Suitable libraries can be prepared from neural tissue samples,e.g., hippocampus and cerebellum tissue, cell lines, and the like. Forexample, the library can be screened with a portion of DNA includingsubstantially the entire receptor subtype-encoding sequence thereof, orthe library may be screened with a suitable oligonucleotide probe basedon a portion of the DNA.

As used herein, a probe is single-stranded DNA or RNA that has asequence of nucleotides that includes at least about 25-30 contiguousbases that are the same as (or the complement of) any 25 or morecontiguous bases set forth in any of SEQ ID Nos. 1, 3, 5, 7, 9 or 11.Preferred regions from which to construct probes include 5' and/or 3'coding sequences, sequences predicted to encode transmembrane domains,sequences predicted to encode cytoplasmic loops, signal sequences,ligand binding sites, and the like.

Either the full-length cDNA clones, fragments thereof, oroligonucleotides based on portions of the cDNA clones can be used asprobes, preferably labeled with suitable label means for readydetection. When fragments are used as probes, DNA sequences for suchprobes will preferably be derived from the carboxyl end-encoding portionof the DNA, and most preferably will include predicted transmembranedomain-encoding portions of the DNA sequence (the domains can bepredicted based on hydropathy analysis of the deduced amino acidsequence using, for example, the method of Kyte and Doolittle (1982), J.Mol. Biol. Vol. 157:105). These probes can be used, for example, for theidentification and isolation of additional members of the glutamatereceptor family.

As a particular application of the invention sequences, geneticscreening can be carried out using the nucleotide sequences of theinvention as probes. Thus, nucleic acid samples from patients havingneuropathological conditions suspected of involvingalteration/modification of any one or more of the glutamate receptorscan be screened with appropriate probes to determine if anyabnormalities exist with respect to any of the endogenous glutamatereceptors. Similarly, patients having a family history of disease statesrelated to glutamate receptor dysfunction can be screened to determineif they are also predisposed to such disease states.

In accordance with another embodiment of the present invention, there isprovided a method for identifying DNA encoding human metabotropicglutamate receptor protein subtypes, said method comprising:

contacting human DNA with a nucleic acid probe as described above,wherein said contacting is carried out under low- to moderate-stringencyhybridization conditions when the probe used is a polynucleic acidfragment, or under high-stringency hybridization conditions when theprobe used is an oligonucleotide, and

identifying DNA(s) which hybridize to said probe.

After screening the library, positive clones are identified by detectinga hybridization signal; the identified clones are characterized byrestriction enzyme mapping and/or DNA sequence analysis, and thenexamined, by comparison with the sequences set forth herein to ascertainwhether they include DNA encoding a complete metabotropic glutamatereceptor subtype (i.e., if they include translation initiation andtermination codons). If the selected clones are incomplete, they may beused to rescreen the same or a different library to obtain overlappingclones. If the library is genomic, then the overlapping clones mayinclude exons and introns. If the library is a cDNA library, then theoverlapping clones will include an open reading frame. In bothinstances, complete clones may be identified by comparison with the DNAand deduced amino acid sequences provided herein.

Complementary DNA clones encoding various human metabotropic glutamatereceptor subtypes (e.g., mGluR1, mGluR2, mGluR3, mGluR5) have beenisolated. Each subtype appears to be encoded by a different gene. TheDNA clones provided herein may be used to isolate genomic clonesencoding each subtype and to isolate any splice variants by screeninglibraries prepared from different neural tissues. Nucleic acidamplification techniques, which are well known in the art, can be usedto locate DNA encoding splice variants of human metabotropic glutamatereceptor subtypes. This is accomplished by employing oligonucleotidesbased on DNA sequences surrounding known or predicted divergentsequence(s) as primers for amplifying human RNA or genomic DNA. Size andsequence determinations of the amplification products can reveal theexistence of splice variants. Furthermore, isolation of human genomicDNA sequences by hybridization can yield DNA containing multiple exons,separated by introns, that correspond to different splice variants oftranscripts encoding human metabotropic glutamate receptor subtypes.

It has been found that not all metabotropic glutamate receptor subtypes(and variants thereof) are expressed in all neural tissues or in allportions of the brain. Thus, in order to isolate cDNA encoding aparticular subtype (or splice variants thereof), it is preferable toscreen libraries prepared from different neuronal or neural tissues orcells. Preferred libraries for obtaining DNA encoding each subtypeinclude: cerebellum to isolate human mGluR1-encoding DNAS; hippocampusto isolate human mGluR2-encoding DNAS; hippocampus and cerebellum toisolate mGluR3-encoding DNAs; hippocampus and cerebellum to isolatemGluR5-encoding DNAs; and the like.

Once DNA encoding a particular receptor subtype has been isolated,ribonuclease (RNase) protection assays can be employed to determinewhich tissues express MRNA encoding such subtype (or splice variantthereof). These assays provide a sensitive means for detecting andquantitating an RNA species in a complex mixture of total cellular RNA.The subtype DNA is labeled and hybridized with cellular RNA. Ifcomplementary mRNA is present in the cellular RNA, a DNA-RNA hybridresults. The RNA sample is then treated with RNase, which degradessingle-stranded RNA. Any RNA-DNA hybrids are protected from RNasedegradation and can be visualized by gel electrophoresis andautoradiography. in situ hybridization techniques can also be used t odetermine which tissues express mRNAs encoding particular metabotropicglutamate receptor subtypes. Thus, labeled subtype DNAs can behybridized to different brain region slices to visualize subtype mRNAexpression.

It appears that the distribution of expression of some humanmetabotropic glutamate receptor subtypes differs from the distributionof such receptors in rat. For example, even though RNA encoding the ratmGluR5 subtype is abundant in rat hippocampus, but is not abundant inrat cerebellum [see, e.g., Abe et al., J. Biol. Chem. 267: 13361-13368(1992)], human mGluR5-encoding CDNAs were successfully obtained fromhuman cerebellum cDNA libraries. Thus, the distribution of somemetabotropic glutamate receptor subtypes in humans and rats appears tobe different.

The above-described nucleotide sequences can be incorporated intovectors for further manipulation. As used herein, vector (or plasmid)refers to discrete elements that are used to introduce heterologous DNAinto cells for either expression or replication thereof. Selection anduse of such vehicles are well within the skill of the artisan.

An expression vector includes vectors capable of expressing DNAs thatare operatively linked with regulatory sequences, such as promoterregions, that are capable of regulating expression of such DNAfragments. Thus, an expression vector refers to a recombinant DNA or RNAconstruct, such as a plasmid, a phage, recombinant virus or other vectorthat, upon introduction into an appropriate host cell, results inexpression of the cloned DNA. Appropriate expression vectors are wellknown to those of skill in the art and include those that are replicablein eukaryotic cells and/or prokaryotic cells and those that remainepisomal or those which integrate into the host cell genome. Presentlypreferred plasmids for expression of invention metabotropic glutamatereceptor subtypes in eukaryotic host cells, particularly mammaliancells, include cytomegalovirus (CMV) promoter-containing vectors such aspCMV-T7-2 and pCMV-T7-3 (see FIG. 1), pcDNA1, and the like, as well asSV40 promoter-containing vectors and MMTV LTR promoter-containingvectors, such as pMMTVT7(+) or pMMTVT7(-) (modified versions of pMAMneo(Clontech, Palo Alto, Calif.), prepared as described herein), and thelike.

As used herein, a promoter region refers to a segment of DNA thatcontrols transcription of DNA to which it is operatively linked. Thepromoter region includes specific sequences that are sufficient for RNApolymerase recognition, binding and transcription initiation. Thisportion of the promoter region is referred to as the promoter. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of RNApolymerase. These sequences may be cis acting or may be responsive totrans acting factors. Promoters, depending upon the nature of theregulation, may be constitutive or regulated. Exemplary promoterscontemplated for use in the practice of the present invention includethe SV40 early promoter, the cytomegalovirus (CMV) promoter, the mousemammary tumor virus (MMTV) steroid-inducible promoter, Moloney murineleukemia virus (MMLV) promoter, and the like.

As used herein, the term "operatively linked" refers to the functionalrelationship of DNA with regulatory and effector sequences ofnucleotides, such as promoters, enhancers, transcriptional andtranslational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. In order to optimize expression and/or in vitro transcription, itmay be necessary to remove, add or alter 5' and/or 3' untranslatedportions of the clones to eliminate extra, potentially inappropriatealternative translation initiation (i.e., start) codons or othersequences that may interfere with or reduce expression, either at thelevel of transcription or translation. Alternatively, consensus ribosomebinding sites (see, for example, Kozak (1991) J. Biol. Chem.266:19867-19870) can be inserted immediately 5' of the start codon andmay enhance expression. Likewise, alternative codons, encoding the sameamino acid, can be substituted for coding sequences of the metabotropicglutamate receptor subunits in order to enhance transcription (e.g., thecodon preference of the host cells can be adopted, the presence of G-Crich domains can be reduced, and the like). Furthermore, for potentiallyenhanced expression of metabotropic glutamate receptor subunits inamphibian oocytes, the subunit coding sequence can optionally beincorporated into an expression construct wherein the 5'- and 3'-ends ofthe coding sequence are contiguous with Xenopus β-globin gene 5' and 3'untranslated sequences, respectively. For example, metabotropicglutamate receptor subunit coding sequences can be incorporated intovector pSP64T (see Krieg and Melton (1984) in Nucleic Acids Research12:7057-7070), a modified form of pSP64 (available from Promega,Madison, Wis.). The coding sequence is inserted between the 5' end ofthe β-globin gene and the 3' untranslated sequences located downstreamof the SP6 promoter. In vitro transcripts can then be generated from theresulting vector. The desirability of (or need for) such modificationsmay be empirically determined.

As used herein, expression refers to the process by which polynucleicacids are transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the polynucleic acid is derived fromgenomic DNA, expression may, if an appropriate eukaryotic host cell ororganism is selected, include splicing of the mRNA.

Particularly preferred base vectors which contain regulatory elementsthat can be linked to human metabotropic receptor-encoding DNAs fortransfection of mammalian cells are cytomegalovirus (CMV) promoter-basedvectors such as pCMV-T7-2 and pCMV-T7-3 (described herein) or pcDNA1(Invitrogen, San Diego, Calif.), MMTV promoter-based vectors such aspMMTVT7(+) or pMMTVT7(-) (as described herein), and SV40 promoter-basedvectors such as PSVP (Clontech, Palo Alto, Calif.).

Full-length DNAs encoding human metabotropic glutamate receptor subtypeshave been inserted into vectors pMMTVT7(+), pMMTVT7(-) pCMV-T7-2 orpCMV-T7-3. pCMV-T7-2 (and pCMV-T7-3) are pUC19-based mammalian cellexpression vectors containing the CMV promoter/enhancer, SV40splice/donor sites located immediately downstream of the promoter, a T7bacteriophage RNA polymerase promoter positioned downstream of thesplice sites, followed by an SV40 polyadenylation signal and apolylinker between the T7 promoter and the polyadenylation signal.Placement of metabotropic glutamate receptor subtype DNA between the CMVpromoter and SV40 polyadenylation signal should provide for constitutiveexpression of the foreign DNA in a mammalian host cell transfected withthe construct.

Vectors pMMTVT7(+) and pMMTVT7(-) were prepared by modifying vectorpMAMneo (Clontech, Palo Alto, Calif.). pMAMneo is a mammalian expressionvector that contains the Rous Sarcoma Virus (RSV) long terminal repeat(LTR) enhancer, linked to the dexamethasone-inducible mouse mammarytumor virus (MMTV)-LTR promoter, followed by SV40 splicing andpolyadenylation sites. pMAMneo also contains the E. coli neo gene forselection of transformants, as well as the β-lactamase gene (encoding aprotein which imparts ampicillin-resistance) for propagation in E. coli.

Vector PMMTVT7(+) can be generated by modification of pMAMneo to removethe neo gene and insert the multiple cloning site and T7 and T3promoters from pBluescript (Stratagene, La Jolla, Calif.). Thus,pMMTVT7(+) contains the RSV-LTR enhancer linked to the MMTV-LTRpromoter, a T7 bacteriophage RNA polymerase promoter positioneddownstream of the MMTV-LTR promoter, a polylinker positioned downstreamof the T7 promoter, a T3 bacteriophage RNA polymerase promoterpositioned downstream of the T7 promoter, and SV40 splicing andpolyadenylation sites positioned downstream of the T3 promoter. Theβ-lactamase gene (encoding a protein which impartsampicillin-resistance) from pMAMneo is retained in pMMTVT7(+), althoughit is incorporated in the reverse orientation relative to theorientation in pMAMneo.

Vector pMMTVT7(-) is identical to pMMTVT7(+) except that the positionsof the T7 and T3 promoters are switched, i.e., the T3 promoter inpMMTVT7(-) is located where the T7 promoter is located in pMMTVT7(+),and the T7 promoter in pMMTVT7(-) is located where the T3 promoter islocated in pMMTVT7(+). Therefore, vectors pMMTVT7(+) and pMMTVT7(-)contain all of the regulatory elements required for expression ofheterologous DNA in a mammalian host cell, wherein the heterologous DNAhas been incorporated into the vectors at the polylinker. In addition,because the T7 and T3 promoters are located on either side of thepolylinker, these plasmids can be used for synthesis of in vitrotranscripts of heterologous DNA that has been subcloned into the vectorsat the polylinker.

For inducible expression of human metabotropic glutamate receptorsubtype-encoding DNA in a mammalian cell, the DNA can be inserted into aplasmid such as pMMTVT7(+) or pMMTVT7(-). These plasmids contain themouse mammary tumor virus (MMTV) LTR promoter for steroid-inducibleexpression of operatively associated foreign DNA. If the host cell doesnot express endogenous glucocorticoid receptors required for uptake ofglucocorticoids (i.e., inducers of the MMTV LTR promoter) into the cell,it is necessary to additionally transfect the cell with DNA encoding theglucocorticoid receptor (ATCC accession no. 67200). For synthesis of invitro transcripts, full-length human DNA clones encoding human mGluR1,mGluR3 and mGluR5 can also be subcloned into pIBI24 (InternationalBiotechnologies, Inc., New Haven, Conn.), pCMV-T7-2 or pCMV-T7-3 (seeFIG. 1), pMMTVT7(+), pMMTVT7(-), pBluescript (Stratagene, La Jolla,Calif.), pGEM7Z (Promega, Madison, Wis.), or the like.

In accordance with another embodiment of the present invention, thereare provided cells containing the above-described polynucleic acids(i.e., DNA or mRNA). Such host cells as bacterial, yeast and mammaliancells can be used for replicating DNA and producing metabotropicglutamate receptor subtype(s). Methods for constructing expressionvectors, preparing in vitro transcripts, transfecting DNA into mammaliancells, injecting oocytes, and performing electrophysiological and otheranalyses for assessing receptor expression and function as describedherein are also described in PCT Application Nos. PCT/US91/05625 andPCT/US92/11090, and in co-pending U.S. application Ser. Nos. 07/563,751and 07/812,254. The subject matter of these documents is herebyincorporated by reference herein in their entirety.

Incorporation of cloned DNA into a suitable expression vector,transfection of eukaryotic cells with a plasmid vector or a combinationof plasmid vectors, each encoding one or more distinct genes or withlinear DNA, and selection of transfected cells are well known in the art(see, e.g., Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press).Heterologous DNA may be introduced into host cells by any method knownto those of skill in the art, such as transfection with a vectorencoding the heterologous DNA by CaPO₄ precipitation (see, e.g., Wigleret al. (1979) Proc. Natl. Acad. Sci. 76:1373-1376). Recombinant cellscan then be cultured under conditions whereby the subtype(s) encoded bythe DNA is (are) expressed. Preferred cells include mammalian cells(e.g., HEK293, CHO and Ltk⁻ cells), yeast cells (e.g., methylotrophicyeast cells, such as Pichia pastoris), bacterial cells (e.g.,Escherichia coli), and the like.

While the DNA provided herein may be expressed in any eukaryotic cell,including yeast cells (such as, for example, P. pastoris (see U.S. Pat.Nos. 4,882,279, 4,837,148, 4,929,555 and 4,855,231), Saccharomycescerevisiae, Candida tropicalis, Hansenula polymorpha, and the like),mammalian expression systems, including commercially available systemsand other such systems known to those of skill in the art which expressG-proteins (either endogenously or recombinantly), for expression of DNAencoding the human metabotropic glutamate receptor subtypes providedherein are presently preferred. Xenopus oocytes are preferred forexpression of in vitro mRNA transcripts of DNA encoding those humanmetabotropic receptor subtypes that are coupled to the PIhydrolysis/Ca⁺⁺ signalling pathways. An endogenous inositol triphosphatesecond messenger-mediated pathway in oocytes permits functionalexpression of human metabotropic receptors in these cells. Oocytesexpressing recombinant human metabotropic receptors respond to agonistsvia the oocyte G-protein-coupled IP₃ generation pathway, whichstimulates release of Ca⁺⁺ from internal stores, and reportedlyactivates a chloride channel that can be detected as a delayedoscillatory current by voltage-clamp recording.

Host cells for functional recombinant expression of human metabotropicreceptors preferably express endogenous or recombinant guaninenucleotide-binding proteins (i.e., G-proteins). G-proteins are a highlyconserved family of membrane-associated proteins composed of α, β and γsubunits. The a subunit, which binds GDP and GTP, differs in differentG-proteins. The attached pair of β and γ subunits may or may not beunique; different α chains may be linked to an identical βγ pair or todifferent pairs [Linder and Gilman, Sci. Am. 267:56-65 (1992)]. Morethan 30 different cDNAs encoding G protein α subunits have been cloned[Simon et al., Science 252:802 (1991)]. Four different β polypeptidesequences are known [Simon et al., Science 252:802 (1991)]. Three offive identified γ cDNAs have been cloned [Hurley et al., PNAS U.S.A.81:6948 (1984); Gautam et al., Science 244:971 (1989); and Gautam etal., PNAS U.S.A. 87:7973 (1990)]. The sequences of a fourth γ cDNA[Kleuss et al., Science 259:832 (1993)] and a fifth γ cDNA [Fisher andAronson, Mol. Cell. Bio. 12:1585 (1992)] have been established, andadditional γ subtypes may exist [Tamir et al., Biochemistry 30:3929(1991)]. G-proteins switch between active and inactive states by guaninenucleotide exchange and GTP hydrolysis. Inactive G protein is stimulatedby a ligand-activated receptor to exchange GDP for GTP. In the activeform, the a subunit, bound to GTP, dissociates from the βγ complex, andthe subunits then interact specifically with cellular effector moleculesto evoke a cellular response. Because different G-proteins can interactwith different effector systems (e.g., phospholipase C, adenyl cyclasesystems) and different receptors, it is useful to investigate differenthost cells for expression of different recombinant human metabotropicreceptor subtypes. Alternatively, host cells can be transfected withG-protein subunit-encoding DNAs for heterologous expression of differingG proteins.

In preferred embodiments, human metabotropic glutamate receptorsubtype-encoding DNA is ligated into a vector, and introduced intosuitable host cells to produce transformed cell lines that express aspecific human metabotropic glutamate receptor subtype, or specificcombinations of subtypes. The resulting cell lines can then be producedin quantity for reproducible quantitative analysis of the effects ofknown or potential drugs on receptor function. In other embodiments,mRNA may be produced by in vitro transcription of DNA encoding eachsubtype. This mRNA, either from a single subtype clone or from acombination of clones, can then be injected into Xenopus oocytes wherethe mRNA directs the synthesis of functional human metabotropicglutamate receptor subtypes. Alternatively, the subtype-encoding DNA canbe directly injected into oocytes for expression of functional humanmetabotropic glutamate receptor subtypes. The transfected mammaliancells or injected oocytes may then be used in the methods of drugscreening provided herein.

Eukaryotic cells in which DNA or RNA may be introduced include any cellsthat are transfectable by such DNA or RNA or into which such DNA or RNAmay be injected and which cells express (endogenously or recombinantly)G-proteins. Preferred cells are those that express little, if any,endogenous metabotropic receptors and can be transiently or stablytransfected and also express invention DNA and RNA. Presently mostpreferred cells are those that can form recombinant or heterologoushuman metabotropic glutamate receptors comprising one or more subtypesencoded by the heterologous DNA. Such cells may be identifiedempirically or selected from among those known to be readily transfectedor injected.

Exemplary cells for introducing DNA include cells of mammalian origin(e.g., COS cells, mouse L cells, Chinese hamster ovary (CHO) cells,human embryonic kidney (HEK) cells, African green monkey cells and othersuch cells known to those of skill in the art), amphibian cells (e.g.,Xenopus laevis oocytes), yeast cells (e.g., Saccharomyces cerevisiae,Pichia pastoris), and the like. Exemplary cells for expressing injectedRNA transcripts include Xenopus laevis oocytes. Cells that are preferredfor transfection of DNA are known to those of skill in the art or may beempirically identified, and include HEK293 (which are available fromATCC under accession #CRL 1573); Ltk⁻ cells (which are available fromATCC under accession #CCL1.3); COS-7 cells (which are available fromATCC under accession #CRL 1651); CHO cells (which are available fromATCC under accession #CRL9618, CCL61 or CRL9096); DG44 cells (dhfr⁻ CHOcells; see, e.g., Urlaub et al. (1986) Cell. Molec. Genet. 12: 555); andBHK cells (see Waechter and Baserga, PNAS U.S.A. 79:1106-1110 (1982);also available from ATCC under accession #CRL10314). Presently preferredcells include CHO cells and HEK293 cells, particularly HEK293 cells thatcan be frozen in liquid nitrogen and then thawed and regrown (forexample, those described in U.S. Pat. No. 5,024,939 to Gorman (see,also, Stillman et al. (1985) Mol. Cell. Biol. 5:2051-2060)), DG44, Ltk⁻cells, and the like. Those of skill in the art recognize that comparisonexperiments should also be carried out with whatever host cells areemployed to determine background levels of glutamate production inducedby the ligand employed, as well as background levels of glutamatepresent in the host cell in the absence of ligand.

DNA may be stably incorporated into cells or may be transientlyexpressed using methods known in the art. Stably transfected mammaliancells may be prepared by transfecting cells with an expression vectorhaving a selectable marker gene (such as, for example, the gene forthymidine kinase, dihydrofolate reductase, neomycin resistance, and thelike), and growing the transfected cells under conditions selective forcells expressing the marker gene. To prepare transient transfectants,mammalian cells are transfected with a reporter gene (such as the E.coli β-galactosidase gene) to monitor transfection efficiency.Selectable marker genes are typically not included in the transienttransfections because the transfectants are typically not grown underselective conditions, and are usually analyzed within a few days aftertransfection.

To produce such stably or transiently transfected cells, the cellsshould be transfected with a sufficient concentration ofsubtype-encoding nucleic acids to form human metabotropic glutamatereceptors indicative of the human subtypes encoded by the heterologousDNA. The precise amounts of DNA encoding the subtypes may be empiricallydetermined and optimized for a particular subtype, cells and assayconditions. Recombinant cells that express metabotropic glutamatereceptors containing subtypes encoded only by the heterologous DNA orRNA are especially preferred.

Heterologous DNA may be maintained in the cell as an episomal element ormay be integrated into chromosomal DNA of the cell. The resultingrecombinant cells may then be cultured or subcultured (or passaged, inthe case of mammalian cells) from such a culture or a subculturethereof. Methods for transfection, injection and culturing recombinantcells are known to the skilled artisan. Similarly, the humanmetabotropic glutamate receptor subtypes may be purified using proteinpurification methods known to those of skill in the art. For example,antibodies or other ligands that specifically bind to one or moresubtypes may be used for affinity purification of a given metabotropicglutamate receptor subtype.

As used herein, heterologous or foreign DNA and RNA are usedinterchangeably and refer to DNA or RNA that does not occur naturally aspart of the genome of the cell in which it is present or to DNA or RNAwhich is found in a location or locations in the genome that differ fromthat in which it occurs in nature. Typically, heterologous or foreignDNA and RNA refers to DNA or RNA that is not endogenous to the host celland has been artificially introduced into the cell. Examples ofheterologous DNA include DNA that encodes a human metabotropic glutamatereceptor subtype, DNA that encodes RNA or proteins that mediate or alterexpression of endogenous DNA by affecting transcription, translation, orother regulatable biochemical processes, and the like. The cell thatexpresses heterologous DNA may contain DNA encoding the same ordifferent expression products. Heterologous DNA need not be expressedand may be integrated into the host cell genome or maintainedepisomally.

Those of skill in the art can readily identify a variety of assays whichcan be used to detect the expression of functional mGluRs. Examplesinclude PI turnover assays [see, e.g., Nakajima et al., J. Biol. Chem.267:2437-2442 (1992) and Example 3.C.2], cAMP assays [see, e.g.,Nakajima et al., supra and Example 3.C.4.], calcium ion flux assays[see, e.g., Ito et al., J. Neurochem. 56:531-540 (1991) and Example3.C.1], cGMP assays [see, e.g., Steiner et al., J. Biol. Chem247:1106-1113 (1972)], arachidonic acid release assays [see, e.g.,Felder et al., J. Biol. Chem. 264:20356-20362 (1989)], and the like. Inaddition, cation-based assays (as described herein) can be employed formonitoring receptor-induced changes in intracellular cyclic nucleotidelevels. Such assays employ host cells expressing cyclic nucleotide-gatedion channels. These channels, which occur in, for example, rodphotoreceptor cells, olfactory cells and bovine kidney cells (see, forexample, Kaupp et al., in Nature 342:762-766 (1989), Dhallen et al., inNature 347:184-187 (1990) and Biel et al., in Proc. Natl. Acad. Sci. USA91:3505-3509 (1994), are permeable to cations upon activation by bindingof cAMP or cGMP. Thus, in the invention assay, host cells expressingendogenous or recombinant cyclic nucleotide-gated channels aretransfected (or injected) with nucleic acids encoding receptorssuspected of influencing cyclic nucleotide levels (e.g., metabotropicglutamate receptor-encoding DNA), and then monitored for changes in theamount of cyclic nucleotide activation of the channels. Measuringchanges in cyclic nucleotide activation of channels allows one toindirectly identify as functional those receptors that cause a change incAMP or cGMP levels when activated. The change in the amount ofactivation of the cyclic nucleotide-gated channels can be determined bymeasuring ion flux through the channel either by electrophysiologicalmeasurement of currents or by measuring a change in intracellular cationlevels (e.g., by fluorescence measurement of intracellular calcium).

In assays of cells expressing receptor species that cause a decrease incyclic nucleotides upon activation (e.g., some metabotropic glutamatereceptors), it may be preferable to expose the cells to agents thatincrease intracellular levels of cyclic nucleotides (e.g., forskolin andIBMX) prior to adding a receptor-activating compound to the cells in theassay.

Host cells suitable for use in the above-described assay include anyhost cells suitable for expression of the receptor being studied (e.g.,L cells, HEK293 cells, CHO, cells or Xenopus oocytes for assays ofmetabotropic glutamate receptors). The cells can be sequentiallytransfected (or injected) with nucleic acids encoding a cyclicnucleotide-gated channel and receptor-encoding nucleic acids, or thecells can be co-transfected with the two nucleic acids. Transient orstable transfection, as described in Examples 3A and 3B, can be carriedout.

Cells transfected (or injected) with cyclic nucleotide-gated channelnucleic acid are incubated (typically for ˜24-48 hours) before testingfor function. The activity of the channels can be assessed usinginside-out membrane patches pulled from the transfected cells (so thatthe concentration of cAMP reaching the cytoplasmic face can becontrolled). The transfectants can also be analyzed by single-cell videoimaging of internal calcium levels ([Ca⁺⁺ ]_(i)). This method allowsanalysis of cyclic nucleotide-gated channel activity by measurement ofintracellular calcium levels, which change with the amount of calciuminflux through the channel, as regulated by cyclic nucleotide activationof the channel. The imaging assay can be conducted essentially asdescribed in Example 3.C.4.b.

The DNA, mRNA, vectors, receptor subtypes, and cells provided hereinpermit production of selected metabotropic glutamate receptor subtypes,as well as antibodies to said receptor subtypes. This provides a meansto prepare synthetic or recombinant receptors and receptor subtypes thatare substantially free of contamination from many other receptorproteins whose presence can interfere with analysis of a singlemetabotropic glutamate receptor subtype. The availability of desiredreceptor subtypes makes it possible to observe the effect of a drugsubstance on a particular receptor subtype or combination ofmetabotropic glutamate receptor subtypes, and to thereby perform initialin vitro screening of the drug substance in a test system that isspecific for humans and specific for a human metabotropic glutamatereceptor subtype or combination of metabotropic glutamate receptorsubtypes. The availability of specific antibodies makes it possible toidentify the subtype combinations expressed in vivo. Such specificcombinations can then be employed as preferred targets in drugscreening.

The ability to screen drug substances in vitro to determine the effectof the drug on specific receptor compositions should permit thedevelopment and screening of receptor subtype-specific ordisease-specific drugs. Also, testing of single receptor subtypes orspecific combinations of various receptor subtypes with a variety ofpotential agonists or antagonists provides additional information withrespect to the function and activity of the individual subtypes andshould lead to the identification and design of compounds that arecapable of very specific interaction with one or more receptor subtypes.The resulting drugs should exhibit fewer unwanted side effects thandrugs identified by screening with cells that express a variety ofreceptor subtypes.

Further in relation to drug development and therapeutic treatment ofvarious disease states, the availability of DNAs encoding humanmetabotropic glutamate receptor subtypes enables identification of anyalterations in such genes (e.g., mutations) which may correlate with theoccurrence of certain disease states. In addition, the creation ofanimal models of such disease states becomes possible, by specificallyintroducing such mutations into synthetic DNA sequences which can thenbe introduced into laboratory animals or in vitro assay systems todetermine the effects thereof.

In another aspect, the invention comprises functional peptide fragments,and functional combinations thereof, encoded by the DNAs of theinvention. Such functional peptide fragments can be produced by thoseskilled in the art, without undue experimentation, by eliminating someor all of the amino acids in the sequence not essential for the peptideto function as a glutamate receptor. A determination of the amino acidsthat are essential for glutamate receptor function is made, for example,by systematic digestion of the DNAs encoding the peptides and/or by theintroduction of deletions into the DNAs. The modified (e.g., deleted ordigested) DNAs are expressed, for example, by transcribing the DNA andthen introducing the resulting mRNA into Xenopus oocytes, wheretranslation of the mRNAs will occur. Functional analysis of the proteinsthus expressed in the oocytes is accomplished by exposing the oocytes toligands known to bind to and functionally activate glutamate receptors,and then monitoring the oocytes to see if endogenous channels are inturn activated. If currents are detected, the fragments are functionalas glutamate receptors.

In accordance with still another embodiment of the present invention,there is provided a method for identifying compounds which bind to humanmetabotropic glutamate receptor subtype(s), said method comprisingemploying receptor proteins of the invention in a competitive bindingassay. Such an assay can accommodate the rapid screening of a largenumber of compounds to determine which compounds, if any, are capable ofdisplacing specifically bound [³ H] glutamate, i.e., binding tometabotropic glutamate receptors. Subsequently, more detailed assays canbe carried out with those compounds found to bind, to further determinewhether such compounds act as modulators, agonists or antagonists ofinvention receptors.

Another application of the binding assay of the invention is the assayof test samples (e.g., biological fluids) for the presence or absence ofreceptors of the present invention. Thus, for example, serum from apatient displaying symptoms related to glutamatergic pathway dysfunctioncan be assayed to determine if the observed symptoms are perhaps causedby over- or under-production of such receptor subtype(s).

The binding assays contemplated by the present invention can be carriedout in a variety of ways, as can readily be identified by those of skillin the art. For example, competitive binding assays can be employed,such as radioreceptor assays, and the like.

In accordance with a further embodiment of the present invention, thereis provided a bioassay for identifying compounds which modulate theactivity of human metabotropic glutamate receptor subtypes of theinvention, said bioassay comprising:

(a) exposing cells containing DNA encoding human metabotropic glutamatereceptor subtype(s), wherein said cells express functional metabotropicglutamate receptors, to at least one compound whose ability to modulatethe activity of said receptors is sought to be determined; andthereafter

(b) monitoring said cells for changes in second messenger activity.

The above-described bioassay enables the identification of agonists,antagonists and allosteric modulators of human metabotropic glutamatereceptors. According to this method, recombinant metabotropic glutamatereceptors are contacted with an "unknown" or test substance (in thefurther presence of a known metabotropic glutamate agonist, whenantagonist activity is being tested), the second messenger activity ofthe known glutamate receptor is monitored subsequent to the contact withthe "unknown" or test substance, and those substances which increase ordecrease the second messenger response of the known glutamatereceptor(s) are identified as functional ligands (i.e., modulators,agonists or antagonists) for human metabotropic glutamate receptors.Second messenger activities which can be monitored include changes inthe concentration of intracellular calcium ions, IP₃, cAMP levels, ormonitoring of arachidonic acid release or activation or inhibition ofion current (when the host cell is an oocyte).

In accordance with a particular embodiment of the present invention,recombinant human metabotropic glutamate receptor-expressing mammaliancells or oocytes can be contacted with a test compound, and themodulating effect(s) thereof can then be evaluated by comparing themetabotropic glutamate receptor-mediated response in the presence andabsence of test compound, or by comparing the metabotropic glutamatereceptor-mediated response of test cells, or control cells (i.e., cellsthat do not express metabotropic glutamate receptors), to the presenceof the compound.

As used herein, a compound or signal that "modulates the activity of ametabotropic glutamate receptor subtype" refers to a compound or signalthat alters the activity of metabotropic glutamate receptors so thatactivity of the metabotropic glutamate receptor is different in thepresence of the compound or signal than in the absence of the compoundor signal. In particular, such compounds or signals include agonists andantagonists. The term agonist refers to a substance or signal, such asglutamate or ACPD, that activates receptor function; and the termantagonist refers to a substance that blocks agonist-induced receptoractivation. Antagonists include competitive and non-competitiveantagonists. A competitive antagonist (or competitive blocker) interactswith or near the site specific for the agonist (e.g., ligand orneurotransmitter) for the same or closely situated site. Anon-competitive antagonist or blocker inactivates the functioning of thereceptor by interacting with a site other than the site that interactswith the agonist.

As understood by those of skill in the art, assay methods foridentifying compounds that modulate human metabotropic glutamatereceptor activity (e.g., agonists and antagonists) generally requirecomparison to a control. One type of a "control" cell or "control"culture is a cell or culture that is treated substantially the same asthe cell or culture exposed to the test compound, except the controlculture is not exposed to test compound. For example, in methods thatuse voltage clamp electrophysiological procedures, the same cell can betested in the presence and absence of test compound, by merely changingthe external solution bathing the cell. Another type of "control" cellor "control" culture may be a cell or a culture of cells which areidentical to the transfected cells, except the cells employed for thecontrol culture do not express the recombinant human metabotropicglutamate receptor subtype(s) expressed in the transfected cells. Inthis situation, the response of test cell to test compound is comparedto the response (or lack of response) of receptor-negative (control)cell to test compound, when cells or cultures of each type of cell areexposed to substantially the same reaction conditions in the presence ofcompound being assayed.

In accordance with yet another embodiment of the present invention, thesecond messenger activity of human metabotropic glutamate receptors canbe modulated by contacting such receptors with an effective amount of atleast one compound identified by the above-described bioassay.

In accordance with yet another embodiment of the present invention,there are provided antibodies generated against the above-describedreceptor proteins. Such antibodies can be employed for studying receptortissue localization, subtype composition, structure of functionaldomains, purification of receptors, as well as in diagnosticapplications, therapeutic applications, and the like. Preferably, fortherapeutic applications, the antibodies employed will be monoclonalantibodies.

The above-described antibodies can be prepared employing standardtechniques, as are well known to those of skill in the art, using theinvention receptor proteins or portions thereof as antigens for antibodyproduction. Both anti-peptide and anti-fusion protein antibodies can beused [see, for example, Bahouth et al. (1991) Trends Pharmacol Sci. vol.12:338-343; Current Protocols in Molecular Biology (Ausubel et al.,eds.) John Wiley and Sons, New York (1989)]. Factors to consider inselecting portions of the metabotropic glutamate receptor subtypes foruse as immunogen (as either a synthetic peptide or a recombinantlyproduced bacterial fusion protein) include antigenicity, accessibility(i.e., extracellular and cytoplasmic domains), uniqueness to theparticular subtype, etc.

The availability of subtype-specific antibodies makes possible theapplication of the technique of immunohistochemistry to monitor thedistribution and expression density of various subtypes (e.g., in normalvs diseased brain tissue). Such antibodies could also be employed fordiagnostic and therapeutic applications.

In accordance with still another embodiment of the present invention,there are provided methods for modulating the ion channel activity ofreceptor(s) of the invention by contacting said receptor(s) with aneffective amount of the above-described antibodies.

The antibodies of the invention can be administered to a subjectemploying standard methods, such as, for example, by intraperitoneal,intramuscular, intravenous, or subcutaneous injection, implant ortransdermal modes of administration, and the like. One of skill in theart can readily determine dose forms, treatment regiments, etc,depending on the mode of administration employed.

In accordance with a still further embodiment of the present invention,there is provided a cation-based bioassay for monitoringreceptor-induced changes in intracellular cyclic nucleotide levels, saidbioassay comprising:

introducing nucleic acids encoding receptors suspected of influencingintracellular cyclic nucleotide levels into host cells expressingendogenous or recombinant cyclic nucleotide-gated channels, and

monitoring changes in the amount of cyclic nucleotide activation of saidcyclic nucleotide-gated channels in the presence and absence of ligandfor said receptor suspected of influencing intracellular cyclicnucleotide levels.

The invention will now be described in greater detail by reference tothe following non-limiting examples.

EXAMPLE 1 Isolation of DNA Encoding Human Metabotropic GlutamateReceptors

A. mGluR5 Receptor cDNA

cDNA Library Screening

RNA isolated from human hippocampus tissue was used as a template forthe synthesis of oligo dt-primed, single-stranded cDNA according tostandard procedures [see, for example, Gubler and Hoffman (1983) Gene25:263-269]. The single-stranded cDNA was converted to double-strandedcDNA, and EcoRI/SnaBI/XhoI adaptors were added to the ends of the cDNAs.The cDNAs were separated by size using agarose gel electrophoresis, andthose that were >2.5 kb were ligated into EcoRI-digested λgt10bacteriophage vectors. The resulting primary human hippocampus cDNAlibrary (˜2×10⁵ recombinants) was screened for hybridization to afragment of the DNA encoding the rat mGluR1 receptor (nucleotides 1 to1723 plus 5' untranslated sequence; see Masu et al. (1991) Nature349:760-765). Hybridization was performed in 5× SSPE, 5× Denhart'ssolution, 50% formamide, 0.2% SDS, 200 μg/ml denatured, sonicatedherring sperm DNA at 42° C. and washes were performed in 1.0× SSPE, 0.2%SDS at 65° C. One hybridizing plaque, METAB1, was identified whichcontains a 3273 bp insert.

To obtain additional human mGluR5-encoding clones, METAB1 wasradiolabeled and used to screen two human cerebellum cDNA librariesprepared as follows. cDNA was synthesized using random primers to primefirst-strand cDNA synthesis from RNA isolated from human cerebellumtissue. The cDNAs were pooled based on length and two libraries weregenerated: one with inserts greater than 2.8 kb in length (i.e., alarge-insert library) and one with inserts 1-2.8 kb in length (i.e., amedium-insert library). The libraries (1×10⁶ recombinants in each) werescreened for hybridization to the METAB1 probe using the samehybridization conditions as used for screening the hippocampus libraryfor hybridization to the rat mGluR1 DNA fragment. Washes were performedin 1× SSPE, 0.2% SDS at 55° C. One hybridizing plaque, METAB2, wasidentified in the large-insert library, whereas four hybridizingplaques, METAB3-METAB6, were identified in the medium-insert library.

In another round of screening for human mGluR5-encoding DNAs, a randomlyprimed human hippocampus cDNA library (2×10⁶ recombinants) containinginserts ranging in size from 1-2 kb and the medium-insert cerebellumcDNA library were screened for hybridization to radiolabeled METAB5using the same conditions as those used in screening the large- andmedium-insert cerebellum libraries with METAB1. Three hybridizingplaques (METAB10-METAB12) were identified in the hippocampus library andfive additional hybridizing plaques (METAB13-METAB17) were identified inanother primary screening of the cerebellum library. Selected plaqueswere purified.

Characterization of Isolated Clones

Characterization of the inserts of the purified plaques by restrictionenzyme mapping and DNA sequence analysis revealed that at least threeapparent splice variants of the human mGluR5 transcript were representedby the isolated clones. Analysis of METAB1 indicated that it contains atranslation initiation codon but no translation termination codon. Thededuced amino acid sequence is ˜70% identical to the rat mGluR1 deducedamino acid sequence, but >90% identical to the rat mGluR5 deduced aminoacid sequence [Abe et al. (1992) J. Biol. Chem. 267:13361-13368].

DNA sequence analysis of METAB5 showed that it overlaps the 3' end ofMETAB1 at the 5' end and continues for an additional 343 nucleotides inthe 3' direction. Comparison of the overlapping regions of METAB1 andMETAB5 revealed that METAB1 contains 96 nucleotides that are not presentin METAB5 (i.e., METAB1 contains a 96-nucleotide insertion relative toMETAB5). METAB5 also does not contain a translation termination codon.The insert of METAB12 overlaps the 3' end of METAB5 at the 5' end,however, and extends farther in the 3' direction to include atranslation termination codon.

DNA sequence analysis of METAB2 showed that the first 869 nucleotides atthe 5' end overlap, and are identical to a portion of the 3' end ofMETAB1; however, the sequences of METAB1 and METAB2 diverge at thebeginning of the 96-nucleotide insertion of METAB1. METAB2 extendsapproximately 2700 nucleotides in the 3' direction and contains aputative translation termination codon 4 nucleotides 3' of the point ofdivergence with METAB1.

Partial DNA sequence analysis of METAB14 indicated that it encodes aportion of another human metabotropic receptor, mGluR1 (see Example1.B.).

Preparation of Full-Length mGluR5 cDNA Constructs

Full-length constructs representing three putative splice variants ofthe human mGluR5 transcript, designated mGluR5a, mGluR5b and mGluR5c,can be generated and incorporated into expression vectors for use inpreparing in vitro transcripts of the cDNAs and/or expression of thecDNAs in mammalian cells. The base expression vector typically used ispCMV-T7-3 or pCMV-T7-2 (see FIG. 1). Plasmid pCMV-T7-3 is a pUC19-basedvector that contains a cytomegalovirus (CMV) promoter/enhancer, SV40splice donor/splice acceptor sites located immediately downstream of thepromoter, a T7 bacteriophage RNA polymerase promoter positioneddownstream of the SV40 splice sites, an SV40 polyadenylation signaldownstream of the T7 promoter, and a polylinker between the T7 promoterand the polyadenylation signal. This vector thus contains all theregulatory elements required for expression of heterologous DNA in amammalian host cell, wherein the heterologous DNA has been incorporatedinto the vector at the polylinker. In addition, because the T7 promoteris located just upstream of the polylinker, this plasmid can be used forsynthesis of in vitro transcripts of heterologous DNA that has beensubcloned into the vector at the polylinker. pCMV-T7-3 and pCMV-T7-2differ only in the orientation of the restriction sites in thepolylinker.

To prepare a full-length mGluR5a construct (see Sequence ID No. 7),portions of clones METAB1, METAB5, and METAB12 were ligated together.Initially, the inserts of METAB1, METAB5 and METAB12 were separatelytransferred from λgt10 as EcoRI fragments into EcoRI-digested pGEM-7Zf(Promega, Madison, Wis.) for ease of manipulation. The pGEM-7Zf vectorcontaining the METAB1 insert was digested with ScaI/NheI to release a3.8 kb fragment containing the 5' half of the ampicillin resistance geneand a 5' portion of the METAB1 insert (nucleotides 1-2724 of Sequence IDNo. 7). The pGEM-7Zf vector containing the insert of METAB5 was digestedwith ScaI/NheI to release a 2.6 kb fragment containing the 3' half ofthe ampicillin resistance gene and a 3' portion of METAB5 (nucleotides2725-3469 of Sequence ID No. 7), and this fragment was ligated with the3.8 kb fragment from the pGEM-7Zf vector containing METAB1 to createpGEM-METAB1+5. pGEM-METAB1+5 was digested with ScaI/NotI to release a4.4 kb fragment containing the 5' half of the ampicillin resistance geneand nucleotides 1-3316 of Sequence ID No. 7. This 4.4 kb fragment wasthen ligated with a 2.6 kb fragment obtained by ScaI/NotI (partial)digestion of the pGEM-7Zf vector containing the METAB12 insert [the 2.6kb fragment contains the 3' half of the ampicillin resistance gene and a3' portion of METAB12 (nucleotides 3317-4085 of Sequence ID No. 7)]. Theresulting vector contained the complete mGluR5a coding sequence inpGEM-7Zf. The full-length mGluR5a cDNA was isolated from the vector asan AatII (blunt-ended)-HindIII fragment and subcloned into NotI(blunt-ended)/HindIII-digested pCMV-T7-3 to generate construct mGluR5a1.

In summary, construct mGluR5a1 contains 369 bp of 5' untranslatedsequence from METAB1 (nucleotides 1-369 of Sequence ID No. 7) and acomplete coding sequence (nucleotides 370-3912 of Sequence ID No. 7) forthe mGluR5a variant of the mGluR5 receptor, as well as 173 bp of 3'untranslated sequence (nucleotides 3913-4085 of Sequence ID No. 7). ThemGluR5a-encoding sequence is operatively linked to the regulatoryelements in pCMV-T7-3 for use in expressing the receptor in mammalianhost cells and for use in generating in vitro transcripts of the DNA tobe expressed in Xenopus oocytes.

Two additional mGluR5a constructs (mGluR5a2 and mGluR5a3) were preparedby modification of the 5' untranslated region of the first mGluR5aconstruct. The above-described mGluR5a construct contains sevenpotentially inappropriate ATG translation initiation codons in the 5'untranslated region that precedes the proposed translation initiationcodon (nucleotides 370 to 372 of Sequence ID No. 7). The mGluR5a1construct was digested with Bal3l to accomplish the following: (1)remove 255 nucleotides of sequence (nucleotides 1-255 of Sequence ID No.7, containing six of the seven upstream ATG triplets), thereby creatingmGluR5a2 and (2) remove 348 nucleotides of sequence (nucleotides 1-348of Sequence ID No. 7, containing all upstream ATG triplets), therebycreating mGluR5a3. Thus, mGluR5a2 is identical to mGluR5a1 except thatit lacks some of the 5' untranslated sequence and thus contains only oneATG triplet upstream of the proposed translation initiation codon.Similarly, mGluR5a3 is identical to mGluR5a1 except that it lacks all ofthe ATG triplets upstream of the proposed translation initiation codonand contains only 21 nucleotides of 5' untranslated sequence.

A third mGluR5a construct, MMTV-hmGluR5a, was prepared for use in MMTVpromoter-regulated expression of mGluR5a as follows. mGluR5a3 wasdigested with XbaI. The 4.1 kb fragment containing the SV40 splicesites, the full-length mGluR5a coding sequence (plus 21 nucleotides of5' untranslated sequence and 173 nucleotides of 3' untranslatedsequence) and the polyadenylation signal was isolated, blunt-ended andligated to a 2 kb EcoRI-NdeI (blunt-ended) fragment of pBR322 to createpBR-hmGluR5. Vector pMAMneo (Clontech, Palo Alto, Calif.), whichcontains the MMTV LTR promoter, and ampicillin and neomycin resistancegenes, was digested with BamHI, to remove the neomycin resistance gene,and allowed to religate. The vector was then digested with EcoRI, andthe fragment containing the ampicillin resistance gene was religatedwith the larger vector fragment in the reverse orientation to createpMAMneo⁻ ampopp. This vector was digested with PstI/NheI, and the 2.3 kbfragment containing a 5' portion of the ampicillin resistance gene andthe MMTV-LTR was isolated. Plasmid pBR-hmGluR5 was digested withPstI/XbaI, and the 5.3 kb fragment containing a 3' portion of theampicillin resistance gene and the mGluR5a sequence (with SV40 splicesites and polyadenylation signal) was ligated with the 2.3 kb Pst/NheIfragment of pMAMneo⁻ ampopp to create MMTV-hmGluR5a.

Thus, pMMTV-hmGluR5a contains the MMTV-LTR followed by SV40 splice sitesin operative linkage with the mGluR5a DNA (containing nucleotides349-4085 of Sequence ID No. 7) followed by a polyadenylation signal.

A fourth mGluR5a construct, pSV-hmGluR5, was prepared for use in SV40promoter-regulated expression of mGluR5a as follows. mGluR5a3 waspartially digested with XhoI, treated with Klenow and allowed toreligate to itself, thereby destroying the XhoI site located 3' of themGluR5a DNA. The plasmid was then digested with ScaI/XhoI, generating afragment containing the SV40 splice sites, the full-length mGluR5acoding sequence (plus 21 nucleotides of 5' untranslated sequence and 173nucleotides of 3' untranslated sequence), the polyadenylation signal anda 3' portion of the ampicillin resistance gene. Plasmid pSVβ (Clontech,Palo Alto, Calif.) was digested with ScaI/XhoI, and the fragmentcontaining a 5' portion of the ampicillin resistance gene and the SV40early promoter was ligated to the ScaI/XhoI fragment containing themGluR5a DNA to create pSV-hmGluR5. Thus, pSV-hmGluR5 contains the SV40early promoter followed by SV40 splice sites in operative linkage withthe mGluR5a DNA (containing nucleotides 349-4085 of Sequence ID No. 7)followed by a polyadenylation signal.

To prepare a full-length mGluR5b construct, an mGluR5a construct(mGluR5al, mGluR5a2 or mGluR5a3) was digested with NheI/PmlI to releasea fragment containing nucleotides 2725-3020 of Sequence ID No. 7. Theremaining vector fragment was then ligated to the NheI/PmlI fragmentisolated from METAB1. The resulting vector, mGluR5b, is identical to themGluR5a construct from which it was prepared, except that it includes a96 bp insertion (nucleotides 3000-3095 of Sequence ID No. 9) locatedbetween nucleotides 2999 and 3000 of Sequence ID No. 7. Sequence ID No.9 is the complete nucleotide sequence of the full-length mGluR5b cDNAprepared from vector mGluR5a1.

To prepare a full-length mGluR5c construct, an mGluR5a construct(mGluR5a1, mGluR5a2 or mGluR5a3) is digested with NheI/HindIII (theHindIII site is present in the polylinker of the pCMV-T7-3 portion ofthe mGluR5a vector) to release a fragment containing nucleotides2725-4085 of Sequence ID No. 7. The remaining vector fragment is thenligated to the NheI/HindIII fragment isolated from METAB2. The resultingfull-length cDNA, mGluR5c (Sequence ID No. 11), is identical to themGluR5a construct from which it was prepared for the first 2630nucleotides of the coding sequence; however, at nucleotide 2631 of thecoding sequence, the coding sequences of mGluR5c and mGluR5a diverge(e.g., beginning at nucleotide 3000 of Sequence ID No. 7) with themGluR5c coding sequence having a guanine nucleotide as nucleotide 2631of the coding sequence followed immediately by a translation terminationcodon (nucleotides 3001-3003 of Sequence ID No. 11).

B. mGluR1 Receptor cDNA

cDNA Library Screening

The medium-insert cerebellum library was screened for hybridization to afragment of the DNA encoding the rat mGluR1 receptor (nucleotides 1 to3031 plus 5' untranslated sequence; see Masu et al. (1991) Nature349:760-765). Hybridization was performed in 5× SSPE, 5× Denhart'ssolution, 50% formamide, 0.2% SDS, 200 μg/ml denatured, sonicatedherring sperm DNA at 42° C. and washes were performed in 1× SSPE, 0.2%SDS at 55° C. Three hybridizing plaques, METAB7-METAB9, were identified.

In a subsequent round of screening, an independent plating of 1×10⁶recombinants of the human medium-insert cerebellum cDNA library wasprobed for additional human mGluR1 clones. This plating was screenedsequentially for hybridization first to a DNA fragment containingnucleotides 1-1256 (plus 5' untranslated sequence) of the rat mGluR1cDNA (i.e., a 5' probe) and then to a DNA fragment containingnucleotides 2075-3310 of the rat mGluR1a cDNA (i.e., a 3' probe) usingthe same hybridization and wash conditions as those used in the previousscreening that identified clones METAB7-METAB9. Three clones (METAB18,METAB21 and METAB22) were identified by hybridization to the 5' probe,and four clones (METAB14, METAB20, METAB32 and METAB35) were identifiedby hybridization to the 3' probe.

The 5' rat mGluR1 fragment was used as a probe to screen thelarge-insert human cerebellum cDNA library for further mGluR1 clones.Hybridization and wash conditions were essentially identical to thoseused in isolating the six mGluR1 clones from the medium-insertcerebellum library(except 20% formamide was used in the hybridizationsolution). Three plaques, METAB58, METAB59 and METAB60, hybridized tothe probe.

Characterization of Isolated Clones

The inserts of the purified plaques were characterized by restrictionenzyme mapping and DNA sequence analysis. METAB58 is ˜2.8 kb andcontains 5' untranslated sequence, a translation initiation codon and˜2.3 kb of coding sequence. The 3' end of METAB58 overlaps the 5' end ofMETAB14. METAB14 extends ˜700 bp in the 3' direction and contains atranslation termination codon. Thus, METAB58 and METAB14 overlap toencode a full-length mGluR1 receptor (see Sequence ID No. 1). The otherclones are also partial mGluR1 cDNAs that contain nucleotide sequencesfrom the portion of the mGluR1 coding sequence located between thetranslation initiation and termination codons.

To determine if additional clones encoding the 3' end of the humanmGluR1 transcript were present in human cDNA libraries, the cDNAs fromthe hippocampus/basal ganglia and cerebellum libraries were subjected tonucleic acid amplification. The 5' primer consisted of nucleotides 2218to 2240 of Sequence ID No. 1 whereas the 3' primer was a degenerateoligonucleotide based on amino acids 890-897 of the rat mGluR1a codingsequence (see Pin et al. (1992) Neurobiology 89:10331-10335). Theproducts of the amplification were analyzed by gel electrophoresis. Asingle product (i.e., a 500 bp fragment) was detected in only thehippocampus/basal ganglia library.

To obtain additional clones representing the 3' end of the mGluR1transcript, the hippocampus and cerebellum cDNA libraries can bescreened (using conditions similar to those used for obtaining humanmGluR1 cDNAs described above) with a fragment from the 3' end of the ratmGluR1a cDNA (e.g., the ˜2 kb NcoI/ClaI fragment of the rat mGluR1acDNA). This probe corresponds to a portion of the 3' region of themGluR1 cDNA that does not appear to be alternatively spliced.Hybridizing clones are then analyzed by restriction mapping and DNAsequence analysis to determine if different 3' ends are represented.

Preparation of Full-Length mGluR1 cDNA Constructs

To prepare a full-length construct encoding the B form of the humanmGluR1 receptor, portions of clones METAB58 and METAB14 are ligated.METAB58 is digested with EcoRI/AccI and the 2459 bp fragment containingnucleotides 154-2612 of Sequence ID No. 1 is isolated. The 704 bpfragment of METAB14 (containing nucleotides 2613-3321 of Sequence IDNo. 1) is isolated by digestion of METAB14 with AccI/XhoI. This fragmentis then ligated to the 2459 bp fragment of METAB58 and toEcoRI/SalI-digested vector pCMV-T7-3. The resulting construct encodinghuman mGluR1B contains 234 nucleotides of 5' untranslated sequence(nucleotides 154-387 of Sequence ID No. 1), the entire mGluR1B codingsequence (nucleotides 388-3108 of Sequence ID No. 1), and 213nucleotides of 3' untranslated sequence (nucleotides 3109-3321 ofSequence ID No. 1). The mGluR1B-encoding sequence is operatively linkedto the regulatory elements in pCMV-T7-3 for expression in mammaliancells.

Several methods can be employed to determine which mGluR5 and mGluR1receptor variants are actually expressed in various human tissues. Forexample, oligonucleotides specific for the nucleotide sequences located5' and 3' of the insertions/deletions (i.e., regions of divergence) ofmGluR transcripts described herein can be used to prime nucleic acidamplifications of RNA isolated from various tissues and/or cDNAlibraries prepared from various tissues. The presence or absence ofamplification products and the sizes of the products indicate whichvariants are expressed in the tissues. The products can also becharacterized more thoroughly by DNA sequence analysis.

RNase protection assays can also be used to determine which varianttranscripts are expressed in various tissues. These assays are asensitive method for detecting and quantitating an RNA species in acomplex mixture of total cellular RNA. A portion of the mGluR DNA islabeled and hybridized with cellular RNA. If complementary MRNA ispresent in the cellular RNA, a DNA-RNA hybrid results. The RNA sample isthen treated with RNase, which degrades single-stranded RNA. Any RNA-DNAhybrids are protected from RNase degradation and can be visualized bygel electrophoresis and autoradiography.

Isolation of genomic clones containing human metabotropicreceptor-encoding sequences by, for example, hybridization to the humanmGluR cDNAs disclosed herein and subsequent characterization of theclones provides further information on possible splice variants of themGluR primary transcripts.

C. mGluR3 Receptor cDNA

cDNA Library Screening

A human hippocampus cDNA library (generated using random primers toprime cDNA synthesis and then selecting cDNAs that were 1.0-2.8 kb forligation to λgt10 vectors) was screened for hybridization to a 500 bpSmaI/XbaI fragment of the rat mGluR2 cDNA and a 3 kb AccI-BamHI fragmentof the rat mGluR3 cDNA [see Tanabe et al. (1992) Neuron 8:169-179].Hybridization was performed in 5× SSPE, 5× Denhart's solution, 50%formamide, 0.2% SDS, 200 μg/ml denatured, sonicated herring sperm DNA at42° C. and washes were performed in 0.5× SSPE, 0.2% SDS at 65° C. Threehybridizing plaques, METAB40, METAB41 and METAB45, were identified.

A portion of the 5' end of METAB45 (i.e., the first 244 bp; nucleotides2634-2877 of Sequence ID No. 5) was then used to screen an amplifiedcerebellum library (generated using random primers to prime cDNAsynthesis and then selecting cDNAs that were >2.8 kb for ligation toλgt10 vectors) and an amplified hippocampus cDNA library (generatedusing random primers to prime cDNA synthesis and then selecting cDNAsthat were >2.0 kb for ligation to λgt10 vectors) for additional mGluR3clones. One million clones from each library were screened.Hybridization and wash conditions were identical to those used inisolating METAB40, METAB41 and METAB45 from the hippocampus library.Three hybridizing plaques were identified in each library: METAB46,METAB49 and METAB50 in the cerebellum library and METAB47, METAB48 andMETAB51B in the hippocampus library.

Characterization of Isolated Clones

The inserts of the purified plaques were characterized by restrictionenzyme mapping and DNA sequence analysis. Each of the isolated clonesare partial cDNAs encoding portions of the human mGluR3 receptor, exceptfor clone METAB40, which encodes a portion of the human mGluR2 receptor(see Example 1.D.). Clones METAB41, METAB45 and METAB47-49 containsequence from the 3' end of the mGluR3 coding sequence as well as atranslation termination codon. Clones METAB46, METAB50 and METAB51Bcontain sequence from the 5' end of the mGluR3 cDNA, including atranslation initiation codon, and varying amounts of 5' untranslatedsequence.

Preparation of Full-Length mGluR3 cDNA Constructs

Four constructs containing the full-length human mGluR3 coding sequencewere prepared by ligating portions of METAB48 and METAB46 or METAB51B.The full-length coding sequence is provided in Sequence ID No. 5(nucleotides 1064-3703). The inserts of clones METAB46 and METAB51B wereseparately subcloned into pCMV-T7-3 as EcoRI fragments. The insert ofclone METAB48 was subcloned as an EcoRI fragment into pCMV-T7-2.

To generate construct mGluR3B, the pCMV-T7-3 plasmid containing theMETAB51B insert was digested with ScaI/BglII, and the 2.6 kb fragmentcontaining the 5' half of the ampicillin resistance gene and a 5'portion of the METAB51B insert (nucleotides 748-1671 of Sequence ID No.5) was isolated. This fragment was ligated to a 4.3 kb fragment isolatedfrom a ScaI/BglII digest of the pCMV-T7-2 plasmid harboring the insertof METAB48 [the 4.3 kb fragment contains the 3' half of the ampicillinresistance gene and a 3' portion of METAB48 (nucleotides 1672-3919 ofSequence ID No. 5)]. The resulting construct, mGluR3B, contains 316nucleotides of 5' untranslated sequence (nucleotides 748-1063 ofSequence ID No. 5), the entire mGluR3 coding sequence (nucleotides1064-3703 of Sequence ID No. 5), and 216 nucleotides of 3' untranslatedsequence (nucleotides 3704-3919 of Sequence ID No. 5). ThemGluR3B-encoding sequence is operatively linked to the regulatoryelements from vectors pCMV-T7-3 and pCMV-T7-2 for expression inmammalian cells.

To generate construct mGluR3C, the pCMV-T7-3 plasmid harboring theinsert of METAB46 was digested with ScaI/BglII and the 3.4 kb fragmentcontaining the 5' half of the ampicillin resistance gene and a 5'portion of METAB46 (nucleotides 1-1671 of Sequence ID No. 5) wasisolated. This fragment was ligated to the same ScaI/BglII fragment ofMETAB48 as was used in construct mGluR3B. The resulting construct,mGluR3C, contains 1063 nucleotides of 5' untranslated sequence(nucleotides 1-1063 of Sequence ID No. 5), the entire mGluR3 codingsequence (nucleotides 1064-3703 of Sequence ID No. 5), and 216nucleotides of 3' untranslated sequence (nucleotides 3704-3919 ofSequence ID No. 5). The mGluR3C-encoding sequence is operatively linkedto the regulatory elements from vectors pCMV-T7-2 and pCMV-T7-3 forexpression in mammalian cells.

Construct mGluR3A was generated by digesting mGluR3C with EcoRV and NotIto remove a fragment containing nucleotides 1-1035 of Sequence ID No. 5,making the NotI site blunt-ended and then allowing the larger vectorfragment to re-ligate. Construct mGluR3A contains 28 nucleotides of 5'untranslated sequence (nucleotides 1036-1063 of Sequence ID No. 5), theentire mGluR3 coding sequence (nucleotides 1064-3703 of Sequence ID No.5) and 216 nucleotides of 3' untranslated sequence (nucleotides3704-3919 of Sequence ID No. 5). The mGluR3A-encoding sequence isoperatively linked to the regulatory elements from vectors pCMV-T7-3 andpCMV-T7-2 for expression in mammalian cells.

To generate construct pSV-hmGluR3C (for use in SV40 promoter-regulatedexpression of mGluR3), the pCMV-T7-3 plasmid harboring the insert ofMETAB46 was digested with ScaI/NotI, and the fragment containing the 3'portion of the ampicillin resistance gene and the entire METAB46 insertwas isolated. Plasmid pSVp was digested with ScaI/NotI, and the fragmentcontaining the 5' portion of the ampicillin resistance gene and the SV40early promoter and splice sites was ligated to the ScaI/NotI fragmentfrom the pCMV-T7-3 vector harboring METAB46 to create pSV-METAB46.Plasmid pSV-METAB46 was digested with ScaI/BglII and the fragmentcontaining the 5' portion of the ampicillin resistance gene, the SV40early promoter and splice sites and a 5' portion of METAB46 (nucleotides1-1671 of Sequence ID No. 5) was isolated. This fragment was ligated tothe same ScaI/BglII fragment of METAB48 as was used in constructsmGluR3B and mGluR3C. The resulting construct, pSV-hmGluR3C, contains theSV40 promoter followed by SV40 splice sites in operative linkage withthe mGluR3 DNA (containing nucleotides 1-3919 of Sequence ID No. 5)followed by a polyadenylation signal.

D. mGluR2 Receptor cDNA

Clone METAB40 was isolated from a human hippocampus cDNA library asdescribed in Example 1.C. The insert cDNA of METAB40 is 1100 bp inlength and encodes the 3' end of a human mGluR2 receptor, including atranslation termination codon and 3' untranslated sequence. The first355 nucleotides of METAB40 are provided in Sequence ID No. 3; the last343 nucleotides of METAB40 (which are all from the 3' untranslatedsequence) are provided in Sequence ID No. 13).

To isolate clones containing DNA representing the 5' portion of themGluR2 transcript, the human hippocampus cDNA library can be screenedfor hybridization to an oligonucleotide corresponding to the 5' end ofMETAB40. Hybridizing plaques are purified and characterized by DNAsequence analysis. Clones that overlap with METAB40 and contain atranslation initiation codon can be ligated to METAB40 at appropriaterestriction sites to generate a full-length mGluR2-encoding cDNAconstruct.

EXAMPLE 2 Expression of Recombinant Human Metabotropic GlutamateReceptors in Oocytes

Xenopus oocytes were injected with in vitro transcripts prepared fromconstructs containing DNA encoding human metabotropic receptors.Electrophysiological measurements of the oocyte transmembrane currentswere made using the two-electrode voltage clamp technique (see e.g.,Stuhmer (1992) Meth. Enzymol. 207:319-339).

A. Preparation of In Vitro Transcripts

Recombinant capped transcripts of metabotropic receptor cDNAs containedin construct mGluR5a3 were synthesized from linearized plasmids usingthe Megascript Kit (Cat. #1334, Ambion, Inc., Austin, Tex.). The mass ofeach synthesized transcript was determined by UV absorbance and theintegrity of each transcript was determined by electrophoresis throughan agarose gel.

B. Electrophysiology

Xenopus oocytes were injected with 10-50 ng of metabotropic receptortranscripts per oocyte. The preparation and injection of oocytes werecarried out as described by Dascal [(1987) Crit. Rev. Biochem.22:317-387]. Two-to-six days following mRNA injection, the oocytes wereexamined using the two-electrode voltage clamp technique. The cells werebathed in Ringer's solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl₂, 10mM HEPES, pH 7.3), and the membrane potential was clamped at -80 to -100mV. Drugs were applied by pipetting 60 μl aliquots of drug-containingsolution directly into the bath. Data were sampled at 2-5 Hz with aLabmaster data acquisition board in PC-386 using AXOTAPE version 1.2(Axon Instruments, Foster City, Calif.) software. Data were exported toa laser printer or plotted using Sigmaplot version 5.0.

Metabotropic receptor-modulating compounds, i.e., 0.001-0.1 μMquisqualate, 0.1-10 μM glutamate and 0.1-300 μM 1S,3R-ACPD(1-amino-cyclopentyl-1,3-dicarboxylic acid), were applied to the bathand the transmembrane currents were recorded. Significant currents weredetected after application of the compounds. Dose-response studies inwhich the currents measured after application of varying amounts of eachcompound were compared revealed that the current magnitude increasedwith increasing concentration of each compound. Analysis of these dataenabled a calculation of EC₅₀ values for each compound which were usedin determining the relative potencies of the compounds.

EXAMPLE 3 Recombinant Expression of Human Metabotropic GlutamateReceptor Subunits in Mammalian Cells

Human embryonic kidney (HEK 293) and Chinese hamster ovary (CHO) cells(i.e, DG44 cells; see Urlaub et al. (1986) Som. Cell. Molec. Genet.12:555) were transfected with DNA encoding human metabotropic receptors.Transfectants were analyzed for expression of metabotropic receptorsusing various assays, e.g., inositol phosphate (IP₁) assays, Ca⁺⁺-sensitive fluorescent indicator-based assays, and [³ H]-glutamatebinding assays.

A. Transient Transfection of HEK 293 Cells

HEK 293 cells were transiently transfected with DNA encoding mGluR5a(constructs mGluR5a2 and mGluR5a3 and construct MMTV-hmGluR5a)receptors. Approximately 2×10⁶ HEK cells were transiently transfectedwith 5-18 μg (or 0.18 μg in some transfections, see Example 3.C.2.) ofthe indicated plasmid according to standard CaPO₄ transfectionprocedures [see Wigler et al. (1979) Proc. Natl. Acad. Sci. USA76:1373-1376]. In addition, 0.5-2 μg (or 0.18 μg in some transfections,see Example 3.C.2) of plasmid pCMVβgal (Clontech Laboratories, PaloAlto, Calif.), which contains the Escherichia coli β-galactosidase genefused to the CMV promoter, were co-transfected as a reporter gene formonitoring the efficiency of transfection. The transfectants wereanalyzed for β-galactosidase expression by direct staining of theproduct of a reaction involving β-galactosidase and the X-gal substrate[Jones (1986) EMBO 5:3133-3142]. Transfectants can also be analyzed forβ-galactosidase expression by measurement of β-galactosidase activity[Miller (1972) in Experiments in Molecular Genetics, pp.352-355, ColdSpring Harbor Press].

HEK 293 cells that were transiently transfected with 5 μg ofMMTV-hmGluR5A were co-transfected with 5 μg of pRShGR (ATCC accessionno. 67200) which contains DNA encoding a glucocorticoid receptoroperatively linked to the Rous Sarcoma virus (RSV) LTR promoter.Co-expression of glucocorticoid receptors in these cells should insurethat induction of expression of the MMTV promoter-mGluR5a DNA occursupon addition of glucocorticoid (e.g., dexamethasone) to the cells.

The efficiency of these transfections of HEK cells was typical ofstandard efficiencies (i.e., ˜50%).

B. Stable Transfection of Mammalian Cells

Mammalian cells, such as HEK 293, Ltk⁻ and CHO cells (e.g., DG44 cells),can be stably transfected using the calcium phosphate transfectionprocedure [Current Protocols in Molecular Biology, Vol. 1, WileyInter-Science, Supplement 14, Unit 9.1.1-9.1.9 (1990)]. When CHO cellsare used as hosts, it is generally preferable to use the SV40 promoterto regulate expression of the human metabotropic receptor-encoding cDNA.Ten-cm plates, each containing 1-2×10⁶ cells, are transfected with 1 mlof DNA/calcium phosphate precipitate containing approximately 5-10 μg ofmetabotropic receptor-encoding DNA and 0.5-1 μg of DNA encoding aselectable marker, for example, the neomycin-resistance gene (i.e.,pSV2neo) for selection of HEK 293 transformants, the thymidine kinasegene for Ltk⁻ cell transfectants, or the dihydrofolate reductase (dhfr)gene for selection of DG44 cell transformants. After ˜14 days of growthin the appropriate selective media, colonies form and are individuallyisolated using cloning cylinders. The isolates are then subjected tolimiting dilution and screened to identify those that expressmetabotropic receptors using, for example, methods described below.

C. Analysis of Transfectants

1. Fluorescent Indicator-Based Assays

Activation of G-protein-coupled metabotropic receptors by agonists leadsto stimulation of the phosphatidylinositol (PI) hydrolysis/intracellularCa⁺⁺ signalling pathway and/or the inhibitory cAMP cascade. Methods ofdetecting transient increases in intracellular calcium concentration canbe applied to the analysis of functional expression of metabotropicreceptors that are coupled to the PI hydrolysis/Ca⁺⁺ mobilizationpathway or to both the PI hydrolysis/Ca⁺⁺ mobilization pathway and theinhibitory cAMP cascade. One method for measuring intracellular calciumlevels relies on calcium-sensitive fluorescent indicators.

Calcium-sensitive indicators, such as fluo-3 and fura-2 (MolecularProbes, Inc., Eugene, Oreg.) are available as acetoxymethyl esters whichare membrane permeable. When the acetoxymethyl ester form of theindicator enters a cell, the ester group is removed by cytosolicesterases, thereby trapping the free indicator in the cytosol.Interaction of the free indicator with calcium results in increasedfluorescence of the indicator; therefore, an increase in theintracellular Ca⁺⁺ concentration of cells containing the indicator canbe expressed directly as an increase in fluorescence (or an increase inthe ratio of the fluorescence at two wavelengths when fura-2 is used).An automated fluorescence detection system for assaying metabotropicreceptors has been described in commonly assigned pending U.S. patentapplication Ser. No. 07/812,254 and corresponding PCT Patent ApplicationNo. US92/11090, both of which are hereby incorporated by referenceherein. Additionally, fluorescence imaging techniques can be utilized tovisualize intracellular Ca⁺⁺ oscillations.

HEK cells th at were transiently transfected with DNA encoding a humanmGluR5a receptor were analyzed for expression of functional recombinantmetabotropic receptors using the automated fluorescent indicator-basedassay and the fluorescence imaging assay. Likewise, cells stablytransfected with metabotropic receptor DNAs can also be analyzed forfunctional metabotropic receptors using these assay systems.

a. Automated Fluorescence Assay

Untransfected HEK 293 cells (or HEK 293 cells transiently transfectedwith PCMV-T7-3) and HEK 293 cells that had been transfected withmGluR5a-encoding DNA were plated in the wells of a 96-well microtiterdish (Nunc Catalog No. 1-6708, distributed by Alameda Industries,Escondido, Calif.) that had been precoated with poly-L-lysine at adensity of 2×10⁵ cells/well and loaded with fluo-3 by incubation for 2hours at 20° C. in a medium containing 20 μM fluo-3, 0.2% Pluronic F-127in HBS (125 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 0.62 mM MgCl₂, 20 mMglucose, 20 mM HEPES, pH 7.4). The cells were then washed with assaybuffer (i.e. HBS). The microtiter dish was then placed into afluorescence plate reader (e.g., Fluoroskan II, Lab ProductsInternational, Ltd., Raleigh, N.C.), and the basal fluorescence of eachwell was measured and recorded before addition of metabotropicreceptor-modulating compounds such as quisqualate, glutamate, trans-ACPD(1-amino-cyclopentane-1,3-dicarboxylic acid), 1S,3R-ACPD, AP3(2-amino-3-phosphonopropionate) AP5 (2-amino-5-phosphonopentanoate), andCNQX (6-cyano-7-nitroquinoxaline-2,3-dione) to the wells. Thefluorescence of the wells was monitored repeatedly (75 readings at0.63-sec intervals) following addition of agonist.

In general, the fluorescence of the untransfected HEK 293 cells did notchange after addition of any of these compounds. The fluorescence of HEK293 cells transiently transfected with either the mGluR5a3 orMMTV-hmGluR5a constructs increased in response to application ofglutamate, quisqualate, trans-ACPD, or 1S,3R-ACPD. The fluorescenceincreased to a peak value, then decreased over time to the basal levelof fluorescence in cells prior to application of the compounds. Theeffects of AP3, AP5 or CNQX on glutamate-, quisqualate- ortrans-ACPD-stimulated fluorescence increases in cells transfected withmGluR5a2 were also investigated. Neither of these compounds (AP3, AP5 orCNQX) inhibited the agonist-induced fluorescence increases in thesecells.

Dose-response studies in which the peak fluorescence values measuredafter application of varying amounts of glutamate, quisqualate or1S,3R-ACPD to cells transfected with mGluR5a3 were compared revealedthat the magnitude of the peak fluorescence increased with increasingconcentration of each compound. Analysis of these data enabled acalculation of EC₅₀ values for each compound which were used indetermining the relative potencies of the compounds.

HEK 293 cells transiently co-transfected with MMTV-hmGluR5a and pRShGR(a glucocorticoid receptor construct) were also analyzed in thefluorescence assay. The fluorescence of these cells increased inresponse to 100 μM quisqualate; the peak response was greater when thecells were preincubated with dexamethasone (˜1 M) for 16 hrs at 37° C.before being assayed.

b. Fluorescence imaging assay

HEK 293 cells that had been transiently transfected with mGluR5a3 anduntransfected HEK 293 cells (control) were analyzed by digital videoimaging in order to visualize metabotropic receptor-mediated changes inintracellular Ca⁺⁺ concentration. Transfectants (4×10⁵ cells per 35-mmculture dish with glass-insert bottom) were loaded with fura-2 byexposing the cells to 1 μM fura-2 (acetoxymethyl ester) for 25 min atroom temperature in the dark. The cells were then washed three timeswith DMEM and four times with Ringer's (160 mM NaCl, 5 mM KCl, 2 mMCaCl₂, 1 mM MgCl₂, 11 mM glucose, 5 mM HEPES, pH 7.3) solution.

The transfectants and untransfected cells were then placed on the stageof an Axiovert 100 TV inverted microscope (Zeiss, Oberkochren, Germany)equipped with a 150 W xenon lamp as the UV light source. An Image 1Fluor System (Universal Imaging, West Chester, Pa.) was used to controlthe alternate excitation of the cells at 340 and 380 nm (typically every3 sec) through a 40× 1.3 N.A. oil immersion objective. Light emitted atgreater than 510 nm was collected by a CCD 72 intensified CCD camera(MTI Dage, Michigan City, Ind.) and digitized. The background emittedlight was subtracted from the 340 and 380 nm excitation images. Thecorrected values were used in calculating the 340/380 intensity ratio.These uncalibrated fura-2 ratio values were reliable indicators ofchanges in the intracellular Ca⁺⁺ concentration.

The uncalibrated fura-2 ratios were used to generate pseudocolor imageswith purple corresponding to resting intracellular Ca⁺⁺ concentration(˜100 nM) and red to high intracellular Ca⁺⁺ concentration (˜1 μM). Forquantitative analysis, the average ratio value in a 12-by-12 pixelregion over each cell was calculated by the software for each ratioimage in an experiment and imported into a spreadsheet for furtheranalysis and graphing.

To demonstrate that HEK 293 cells express the intracellular componentsrequired in receptor-mediated activation of the PI hydrolysis/Ca⁺⁺mobilization pathway, transfectants and untransfected cells (whichexpress endogenous G-protein-coupled muscarinic acetylcholine receptors)were exposed to 1 mM carbamylcholine (CCh; a muscarinic acetylcholinereceptor agonist), and the cells were monitored for increases inintracellular Ca⁺⁺ concentration. Typically, a detectable increase inthe intracellular Ca⁺⁺ concentration of the majority of the cells wasobserved in response to CCh addition in the imaging studies.

Both transfected and untransfected HEK 293 cells were also monitored forincreases in intracellular Ca⁺⁺ concentration in response to 100 μMquisqualate. On average, the intracellular Ca⁺⁺ concentration of theuntransfected cells did not change after exposure to quisqualate. Incontrast, the intracellular Ca⁺⁺ concentration of 26.7±22.3% of thetransfected cells increased in response to application of 100 μMquisqualate.

2. Phosphatidylinositol Hydrolyis (IP₁) Assays

Because activation of G-protein-coupled metabotropic receptors byagonists can lead to stimulation of the phosphatidylinositol (PI)hydrolysis pathway, methods of detecting increases in the products of PIhydrolysis (e.g., IP₃, IP₂ or IP₁) can be applied to the analysis offunctional expression of metabotropic receptors that are coupled to thePI hydrolysis/Ca⁺⁺ mobilization pathway or to both the PIhydrolysis/Ca⁺⁺ mobilization pathway and the inhibitory cAMP cascade.One method for measuring IP₁ and/or IP₂ and/or IP₃ generated byhydrolysis of PI involves incorporation of [³ H]-myo-inositol into cellmembrane phospholipids and subsequent separation of [³ H]-IP₁, [³ H]-IP₂and [³ H]-IP₃, followed by quantitation of the radioactivity in eachfraction, as follows.

HEK 293 cells that had been transiently transfected with mGluR5a3 wereplated in 24-well microtiter plates at a density of 8×10⁵ cells/well.After the cells were allowed to settle and adhere to the bottom of theplate for a few hours, 2 μCi of [³ H]-myo-inositol (Amersham catalog #PT6-271, Arlington Heights, Ill.; specific activity=17.7 Ci/mmol) wasadded to each well and incubated overnight at 37° C. The next day, thecells were examined under a Nikon Diaphot inverted microscope to assessthe health of the cells morphologically as well as to determine if thewells contained a confluent layer of cells. Media was then aspirated andthe cells were washed twice with 0.5 ml Krebs bicarbonate buffer [117.9mM NaCl, 4.72 mM KCl, 2.54 mM CaCl₂, 1.18 mM MgSO₄, 1.19 mM KH₂ PO₄, 25mM NaHCO₃, 11.1 mM dextrose (equilibrated with 95% O₂, 5% CO₂, pH 7.4)].The cells were incubated for 45 min. at room temperature. The buffer wasthen aspirated from each well and the cells were washed and incubated in0.5 ml/well for 45 min at room temperature. The buffer was aspiratedfrom each well, and the cells were then incubated for 20 min at 37° C.with 450 μl Krebs-bicarbonate buffer containing 10 mM LiCl instead of 10mM NaCl (to block hydrolysis of IP₁ to inositol and inorganic phosphate)and 10 mM unlabeled myo-inositol.

To begin treatment of the cells with metabotropic receptor-modulatingcompounds, 50 μl of Krebs-bicarbonate buffer (control) or 10× the finalconcentration of the compound was added to each well and the incubationwas continued for 40 min. Incubation was terminated by addition of 1 mlice-cold methanol to each well.

In order to isolate IP₁ from the cells, the cells were removed from theplates by scraping with plastic pipette tips, and the cell suspensionwas transferred to 12×75 mm glass tubes. The tubes were thoroughlyvortexed, and a 150-μl aliquot, i.e., one-tenth of the total volume, ofeach reaction mixture was transferred to another tube for proteindetermination. The water-soluble inositol phosphates were separated fromthe radiolabelled membrane phospholipids by extraction in 1 mlchloroform. The tubes were incubated at room temperature for 30 minbefore centrifugation at 500×g for 5 min at 4° C. The aqueous (top)layer containing the [³ H]-inositol phosphates was transferred to 10-mlsyringes connected to Accell QMA SEP-PAK columns (Millipore;California), which were attached to an Amersham Superseparator apparatusthat was modified to allow collection into 20-ml scintillation vials.Water (10 ml) was added to the cartridge to remove [³ H]-inositolprecursor, followed by 4 ml 0.02 M triethylammonium hydrogen carbonatedbuffer (TEAB, Fluka; New York). To separately remove [³ H]-IP₁, [³H]-IP₂ and [³ H]-IP₃ from the cartridge, 4 ml of 0.1 M TEAB, 4 ml of 0.3M TEAB and 4 ml of 0.4 M TEAB were sequentially added to the cartridgeand the separate eluate fractions were collected in large scintillationvials. Ecolume cocktail (15 ml; ICN; California) was added to each vialfor subsequent scintillation counting to determine the amount of each IPin the separate fractions. Protein concentration was determined usingthe Bio-Rad Protein Micro-Assay (Bio-Rad, Richmond, Calif.).

HEK 293 cells transiently transfected with 18 μg of mGluR5a3 displayedrelatively high basal levels of IP₁ when analyzed in this assay.However, HEK 293 cells transiently transfected with 0.18 μg of mGluR5a3exhibited lower basal IP₁ levels and detectable increases in IP₁ levelswhen treated with 1 mM glutamate, 1 mM quisqualate or 1 mM 1S,3R-ACPD.The quisqualate-induced increase in IP₁ levels was not affected by 1 mMAP3.

Dose-response studies which compared the IP₁ levels measured afterapplication of varying amounts of glutamate, quisqualate or 1S,3R-ACPDto cells transfected with mGluR5a3 revealed that IP₁ levels increasedwith increasing concentration of each compound. Analysis of these dataenabled calculation of EC₅₀ values for each compound which were used indetermining the relative potencies of the compounds.

3. Metabotropic Receptor Ligand Binding Assays

HEK cells transiently transfected with mGluR5a3 or with pUC19 (negativecontrol) were analyzed for [³ H]-glutamate binding. Rat brain membraneswere included in the binding assays as a positive control.

a. Preparation of Membranes

i. Rat Forebrain Membranes

Rat forebrain membranes were prepared from rat brains as described bySchoepp et al. [(1992) Neurosci. Lett. 145:100]. Briefly, forebrains,consisting essentially of cerebral cortex, striatum and hippocampus,from ten rat brains were homogenized in 50 volumes of 30 mM ice-coldTris-HCl containing 2.5 mM CaCl₂, pH 7.6 using a Polytron (Brinkman,Westbury, N.Y.). The homogenate was centrifuged at 30,000×g for 15minutes at 4° C. The supernatant was discarded, the pellet wasresuspended in 50 volumes of buffer using a Polytron and the suspensionwas centrifuged at 30,000×g for 15 min. This step was repeated twice.The pellet was resuspended in buffer and incubated at 37° C. for 30 min.The suspension was then centrifuged at 30,000×g for 15 min. at 4° C.This step was repeated three times. The final pellet was resuspended in15 volumes of 50 mM Tris-HCl, pH 7.6, buffer, aliquoted, quick frozenand stored at -70° C.

ii. Membranes from Transfected and Untransfected HEK293 Cells

In order to prepare membranes from HEK 293 cells transfected withmGluR5a-encoding DNA or pUC19 (negative control), cells were scrapedfrom the tissue culture plates, and the plates rinsed with 5 ml of PBS(phosphate-buffered saline: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂ HPO₄, 1.7mM KH₂ PO₄). The cells were centrifuged at low speed in a table-topcentrifuge, and the cell pellet was rinsed with PBS. The cell pellet wasresuspended in 20 volumes of 50 mM Tris-HCl containing 0.5 mM PMSF, pH7.6. The cells were homogenized on ice in a Dounce (teflon/glass)homogenizer using 10-20 strokes. The homogenate was centrifuged at12,000×g for 30 min. at 4° C. The final membrane pellet was resuspendedin 50 mM Tris-HCl containing 0.5 mM PMSF, pH 7.6. The membranepreparations were aliquoted, quick-frozen, and stored at -70° C. Theprotein concentration was determined using the method of Bradford[(1976) Anal. Biochem. 72:248].

b. [³ H]-Glutamate Binding Assays

Specific binding of [³ H]-glutamate to metabotropic receptors in ratforebrain membranes was determined basically as described by Schoepp etal. (supra). On the day of the assay, frozen homogenate was thawed andwashed three times with 50 mM Tris-HCl, pH 7.6. The final pellet wasresuspended in 50 mM Tris-HCl, pH 7.6. The protein concentration wasdetermined using the method of Bradford [(1976) Anal. Biochem. 72:248].The suspension was centrifuged at 30,000×g for 15 min. in order to beable to resuspend the pellet in the assay buffer (50 mM Tris-HCl, 0.5 mMPMSF, 0.1% BSA, pH 7.6) at a concentration of 1 mg/ml. The membranesuspension was incubated in triplicate with 10 or 100 nM [³ H]-glutamate(New England Nuclear, Boston, Mass.; catalog no. NET-490, specificactivity=57.4 Ci/mmol) in a total volume of 0.5 ml assay buffercontaining 100 μM NMDA (Sigma, St. Louis, Mo.), 100 μM AMPA and 100 μMkainate (Research Biochemicals Inc., Natick, Mass.) to block [³H]-glutamate binding to ionotropic glutamate receptors and 100 μM SITS(Sigma, St. Louis, Mo.) to inhibit [³ H]-glutamate binding tochloride-dependent uptake sites for 45 min on ice. Bound radioactivitywas separated from free radioactivity by centrifugation for 5 min. at20,000×g (4° C.) in an SM-24 rotor (Sorvall, Wilmington, Del.). Thepellets were washed twice with 5-6 ml of ice-cold 50 mM Tris-HCl buffer,pH 7.6. The pellets were solubilized by vortexing in 5 ml of Ecolumescintillation cocktail. The radioactivity was measured in a Beckmanscintillation counter. The nonspecific binding observed in the presenceof 1 mM glutamate was subtracted from the total binding in order todetermine specific binding.

Specific binding of [³ H]-glutamate to membranes prepared from HEK 293cells transfected with mGluR5a-encoding DNA or pUC19 was determinedessentially as described for measuring binding to rat brain membraneswith minor modifications. On the day of the assay, frozen homogenate wasthawed and centrifuged in a MR-150 high-speed refrigeratedmicrocentrifuge (Peninsula Laboratories, Inc., Belmont, Calif.). Thepellet was washed twice with assay buffer (50 mM Tris-HCl, 0.5 mM PMSF,0.1% BSA, pH 7.6), and the final pellet was resuspended in assay bufferat a concentration of 1 mg/ml. NMDA, AMPA and kainate were excluded fromthe assay mixture when HEK 293 cell membranes were being analyzed for [³H]-glutamate binding.

Specific binding of [³ H]-glutamate to rat brain membranes was measuredusing 200 μg of membrane and 100 nM [³ H]-glutamate. The ratio oftotal-to-nonspecific binding was approximately 2:1.

Specific binding of [³ H]-glutamate to membranes prepared from HEK 293cells transfected with mGluR5a3 or pUC19 was measured using 200 μg ofmembranes and 100 nM [³ H]-glutamate. The amount of specific binding tomembranes prepared from HEK 293 cells transfected with mGluR5a3 wassignificantly higher than that to membranes prepared from HEK 293 cellstransfected with pUC19. Competitive binding studies were conducted inwhich the amount of specific binding of [³ H]-glutamate to membranesprepared from HEK 293 cells transfected with mGluR5a3 in the presence ofvarious concentrations of unlabeled glutamate was determined. IC₅₀values were calculated from the data obtained in these studies.

4. Cyclic AMP (cAMP) Assays

a. RIA-Based Assays

Because activation of some G-protein-coupled receptors results indecreases (as opposed to increases) in cAMP, assays that measureintracellular cAMP levels can also be used to evaluate recombinant humanmetabotropic receptors expressed in mammalian host cells. Mammaliancells transiently or stably transfected with human metabotropicreceptor-encoding DNA or pUC19 (negative control) are plated in 24-wellmicrotiter plates at a density of 5×10⁵ cells/well and allowed toincubate overnight. The following day, cells are examined under a NikonDiaphot inverted microscope to assess the health of the cellsmorphologically as well as to determine if the wells contain a confluentlayer of cells. Media is then aspirated and the cells are washed twicewith 0.5 ml Krebs bicarbonate buffer (same buffer used in the PIhydrolysis assay; see Example 3.C.2) containing 1 mM IBMX(3-isobutyl-1-methylxanthine; Sigma, St. Louis, Mo.) and 0.1% BSA.Alternatively, 1× PBS can be used in place of Krebs bicarbonate buffer.Each wash is followed with a 30-min incubation at 37° C. The buffer isaspirated from each well and the cells are then incubated for 20 min at37° C. with 0.2 ml Krebs-bicarbonate buffer containing 1 mM IBMX and0.1% BSA.

To begin treatment of the cells with metabotropic receptor-modulatingcompounds, 50 μl of Krebs-bicarbonate buffer, with or without 5× thefinal concentration of forskolin, is added to some of the cells (basalcontrol) and 5× the final concentration of the compound plus 5× thefinal concentration of forskolin is added to some cells (test cells) andthe incubation is continued for 15 min at 37° C. At the end of this15-min period, the reaction is terminated by adding 25 μl of 1% TritonX-100 solution and the incubation is continued for another 10 min. Thelysed cells plus the cell suspension are transferred to 12×75 mmpolypropylene tubes with plastic pipette tips. Each well is rinsed with75 μl of Krebs-bicarbonate buffer containing 1 mM IBMX and 0.1% BSA. Therinse is combined with the cell lysate. The cell lysate suspension iscentrifuged at 2300×g for 5 min and the supernatant is assayed for cAMPlevels using an RIA kit (Amersham Life Sciences catalog #TRK 432;Arlington Heights, Ill.).

b. Cyclic Nucleotide-Gated Channel-Based Assay

HEK293 cells were grown in monolayers (approximately 2×10⁶ cells per 10cm poly-D-lysine-coated plate) in Dulbecco's modified Eagle's medium(DMEM; Gibco) containing 5% defined supplemented calf serum (Hyclone)including 100 U/ml penicillin and 100 μg/ml streptomycin sulfate. Thecells were transiently transfected by the calcium phosphate method (seeAusubel, et al., supra, pp 9.1.1-9.1.7) with 5 μg of pCMV-OCNA(containing DNA encoding the olfactory cyclic nucleotide-gated channel(see Dhallen et al., supra) linked to the CMV promoter, 2 μg pCMV-βgal(Clontech, Palo Alto, Calif.), and 13 μg pUC19 as a control plasmid.Vector PCMV-OCNA was constructed by isolating the olfactory cyclicnucleotide-gated channel-encoding DNA as ˜3.0 kb EcoRI fragment frompBluescript KS and ligating the resulting fragment to EcoRI-digestedpCMV-T7-3. Six hours after transfection, the calcium phosphateprecipitate was washed off and cells fed with DMEM containing 10%dialyzed fetal bovine serum (Hyclone), 100 U/ml penicillin, 100 μg/mlstreptomycin, and supplemented with 2 mM glutamine. Transfectionefficiencies, as determined by measuring β-galactosidase activity, were50-70%.

HEK cells transfected with olfactory cyclic nucleotide-gated channel DNAwere incubated 24-48 hours before testing for function. The activity ofthe channels was first assessed electrophysiologically using inside-outmembrane patches pulled from the transfected cells so that theconcentration of cAMP reaching the cytoplasmic face could be controlled(see, e.g., Single-Channel Recording, Sakmann and Neher, eds., PlenumPress, N.Y. (1983)). The patch was exposed to Ca⁺⁺ /Mg⁺⁺ -free Ringer'ssolution on both surfaces. In one patch, a current was elicited byramping the membrane potential from -100 to +100 mV in 2 seconds, in thepresence of 1 mM cAMP. This result suggested that the channel wasfunctionally expressed.

The transfectants were also analyzed by single-cell video imaging ofinternal calcium levels ([Ca⁺⁺ ]_(i)). This method allows analysis ofcyclic nucleotide-gated channel activity by measurement of intracellularcalcium levels, which change with the amount of calcium influx throughthe channel, as regulated by cyclic nucleotide activation of thechannel. The imaging assay was conducted essentially as described inExample 3.C.1.b., with some modifications. After dye loading, the cellswere examined using a Zeiss Axiovert microscope and 100 W mercury lamp,a Dage intensified CCD camera, and Image-1 hardware and software forimage processing. The software controlled the alternate excitation ofthe cells at 350 and 385 nm (typically every 5 seconds) through a 20×1.3N.A. oil immersion objective. Light emitted at greater than 510 nm wascollected by the CCD camera, digitized, and 350 and 385 nm excitationimages were background-subtracted before calculating the 350/385 nmintensity ratio.

For quantitative analysis, the average 350/385 ratio value in a 12 by 12pixel region over each cell was calculated by the software for eachratio image in an experiment and imported into a spreadsheet for furtheranalysis and graphing. Fura-2 signals were calibrated with an intactcell in which R_(min) was obtained by exposing the cells to Ringer'ssolution containing 10 μM ionomycin, 10 mM EGTA and no added Ca⁺⁺.R_(max) was next obtained by exposing the cells to Ringer's solutioncontaining 10 μM ionomycin and 10 mM Ca⁺⁺, with three washes. Using aK_(d) of 250 nM for fura-2 inside living cells and the equation ofGrynkiewicz et al. (J. Biol. Chem. 260:3440 (1985)), the resting [Ca⁺⁺]_(i) was typically 100 nM.

In these experiments, the HEK293 cell transfectants were exposed toagents which increase intracellular cAMP levels and monitored forsubsequent changes in [Ca⁺⁺ ]_(i). There was a small increase in [Ca⁺⁺]_(i) in the averaged results from 64 cells, and in individual cells inresponse to addition of 100 μM forskolin (activator of adenyl cyclase).A more significant increase was observed after addition of 1 mM IBMX(inhibitor of cAMP phosphodiesterase). In a control experiment, only 1out of 64 untransfected HEK293 cells showed an increase in [Ca⁺⁺ ]_(i)in response to elevation of intracellular cAMP levels. This response wastransient and clearly different from the sustained response seen inHEK293 cells transfected with the cyclic nucleotide-gated channel DNA.

These results demonstrate that HEK cells expressing cyclicnucleotide-gated channels may be used as host cells in assays ofreceptors that cause a change in intracellular cyclic nucleotide levelswhen activated (e.g., metabotropic receptors).

5. Northern Blot Hybridization Analysis

Cells transfected with human metabotropic receptor-encoding DNA can alsobe analyzed for expression of the corresponding transcript by northernblot analysis. Total RNA was isolated from ˜1×10⁷ cells that have beentransfected with the human metabotropic receptor-encoding DNA, and 10-15μg of RNA is used for northern hybridization analysis. The inserts fromhuman metabotropic receptor-encoding plasmids are nick-translated andused as probes. Typical conditions for northern blot hybridization andwashing are as follows:

hybridization in 5× SSPE, 5× Denhart's solution, 50% formamide, at 42°C. followed by washing in 0.2× SSPE, 0.1% SDS, at 65° C.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

SUMMARY OF SEQUENCES

Sequence ID No. 1 is the nucleic acid sequence (and the deduced aminoacid sequence) of a DNA encoding a metabotropic glutamate receptorsubtype (mGluR1B) of the present invention.

Sequence ID No. 2 is the deduced amino acid sequence of the nucleotidesequence of Sequence ID No. 1.

Sequence ID No. 3 is a nucleotide sequence (and the deduced amino acidsequence) of a partial clone encoding a portion of an human mGluR2receptor subtype.

Sequence ID No. 4 is the amino acid sequence of a portion of an humanmGluR2 receptor subunit as encoded by the nucleotide sequence ofSequence ID No. 3.

Sequence ID No. 5 is the nucleic acid sequence (and the deduced aminoacid sequence) of a DNA encoding a metabotropic glutamate receptorsubtype (mGluR3) of the present invention.

Sequence ID No. 6 is the deduced amino acid sequence of the nucleotidesequence of Sequence ID No. 5.

Sequence ID No. 7 is the nucleic acid sequence (and the deduced aminoacid sequence) of a DNA encoding a metabotropic glutamate receptor(mGluR5a1) of the present invention.

Sequence ID No. 8 is the deduced amino acid sequence of the nucleotidesequence of Sequence ID No. 7.

Sequence ID No. 9 is the nucleic acid sequence (and the deduced aminoacid sequence) of a DNA encoding an mGluR5 variant metabotropicglutamate receptor (mGluR5b) of the present invention.

Sequence ID No. 10 is the deduced amino acid sequence of the nucleotidesequence of Sequence ID No. 9.

Sequence ID No. 11 is the nucleic acid sequence (and the deduced aminoacid sequence) of a DNA encoding an mGluR5 variant metabotropicglutamate receptor (mGluR5c) of the present invention.

Sequence ID No. 12 is the deduced amino acid sequence of the nucleotidesequence of Sequence ID No. 11.

Sequence ID No. 13 is 343 nucleotides of 3' untranslated sequence of anhuman mGluR2 receptor subtype.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 13                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3321 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                   - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 388..3108                                                       (D) OTHER INFORMATION: - #/product= "HUMAN MGLUR1B"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GCCGAGCGTG GCCACGGYCC TCTGGCCCCG GGACCATAGC GCTGTCTACC CC -             #GACTCAGG     60                                                                 - - TACTCAGCAT CTAGCTCACC GCTGCCAACA CGACTTCCAC TGTACTCTTG AT -            #CAATTTAC    120                                                                 - - CTTGATGCAC TACCGGTGAA GAACGGGGAC TCGAATTCCC TTACAAACGC CT -            #CCAGCTTG    180                                                                 - - TAGAGGCGGT CGTGGAGGAC CCAGAGGAGG AGACGAAGGG GAAGGAGGCG GT -            #GGTGGAGG    240                                                                 - - AGGCAAAGGC CTTGGACGAC CATTGTTGGC GAGGGGCACC ACTCCGGGAG AG -            #GCGGCGCT    300                                                                 - - GGGCGTCTTG GGGGTGCGCG CCGGGAGCCT GCAGCGGGAC CAGCGTGGGA AC -            #GCGGCTGG    360                                                                 - - CAGGCTGTGG ACCTCGTCCT CACCACC ATG GTC GGG CTC CTT - #TTG TTT TTT            411                                                                                         - #            Met Val Gly Le - #u Leu Leu Phe Phe                            - #              1    - #           5                        - - TTC CCA GCG ATC TTT TTG GAG GTG TCC CTT CT - #C CCC AGA AGC CCC GGC          459                                                                       Phe Pro Ala Ile Phe Leu Glu Val Ser Leu Le - #u Pro Arg Ser Pro Gly                10             - #     15             - #     20                          - - AGG AAA GTG TTG CTG GCA GGA GCG TCG TCT CA - #G CGC TCG GTG GCC AGA          507                                                                       Arg Lys Val Leu Leu Ala Gly Ala Ser Ser Gl - #n Arg Ser Val Ala Arg            25                 - # 30                 - # 35                 - # 40       - - ATG GAC GGA GAT GTC ATC ATT GGA GCC CTC TT - #C TCA GTC CAT CAC CAG          555                                                                       Met Asp Gly Asp Val Ile Ile Gly Ala Leu Ph - #e Ser Val His His Gln                            45 - #                 50 - #                 55              - - CCT CCG GCC GAG AAA GTG CCC GAG AGG AAG TG - #T GGG GAG ATC AGG GAG          603                                                                       Pro Pro Ala Glu Lys Val Pro Glu Arg Lys Cy - #s Gly Glu Ile Arg Glu                        60     - #             65     - #             70                  - - CAG TAT GGC ATC CAG AGG GTG GAG GCC ATG TT - #C CAC ACG TTG GAT AAG          651                                                                       Gln Tyr Gly Ile Gln Arg Val Glu Ala Met Ph - #e His Thr Leu Asp Lys                    75         - #         80         - #         85                      - - ATC AAC GCG GAC CCG GTC CTC CTG CCC AAC AT - #C ACC CTG GGC AGT GAG          699                                                                       Ile Asn Ala Asp Pro Val Leu Leu Pro Asn Il - #e Thr Leu Gly Ser Glu                90             - #     95             - #    100                          - - ATC CGG GAC TCC TGC TGG CAC TCT TCC GTG GC - #T CTG GAA CAG AGC ATT          747                                                                       Ile Arg Asp Ser Cys Trp His Ser Ser Val Al - #a Leu Glu Gln Ser Ile           105                 1 - #10                 1 - #15                 1 -      #20                                                                              - - GAG TTC ATT AGG GAC TCT CTG ATT TCC ATT CG - #A GAT GAG AAG GAT        GGG      795                                                                    Glu Phe Ile Arg Asp Ser Leu Ile Ser Ile Ar - #g Asp Glu Lys Asp Gly                          125  - #               130  - #               135              - - ATC AAC CGG TGT CTG CCT GAC GGC CAG TCC CT - #C CCC CCA GGC AGG ACT          843                                                                       Ile Asn Arg Cys Leu Pro Asp Gly Gln Ser Le - #u Pro Pro Gly Arg Thr                       140      - #           145      - #           150                  - - AAG AAG CCC ATT GCG GGA GTG ATC GGT CCC GG - #C TCC AGC TCT GTA GCC          891                                                                       Lys Lys Pro Ile Ala Gly Val Ile Gly Pro Gl - #y Ser Ser Ser Val Ala                   155          - #       160          - #       165                      - - ATT CAA GTG CAG AAC CTG CTC CAG CTC TTC GA - #C ATC CCC CAG ATC GCT          939                                                                       Ile Gln Val Gln Asn Leu Leu Gln Leu Phe As - #p Ile Pro Gln Ile Ala               170              - #   175              - #   180                          - - TAT TCA GCC ACA AGC ATC GAC CTG AGT GAC AA - #A ACT TTG TAC AAA TAC          987                                                                       Tyr Ser Ala Thr Ser Ile Asp Leu Ser Asp Ly - #s Thr Leu Tyr Lys Tyr           185                 1 - #90                 1 - #95                 2 -      #00                                                                              - - TTC CTG AGG GTT GTC CCT TCT GAC ACT TTG CA - #G GCA AGG GCC ATG        CTT     1035                                                                    Phe Leu Arg Val Val Pro Ser Asp Thr Leu Gl - #n Ala Arg Ala Met Leu                          205  - #               210  - #               215              - - GAC ATA GTC AAA CGT TAC AAT TGG ACC TAT GT - #C TCT GCA GTC CAC ACG         1083                                                                       Asp Ile Val Lys Arg Tyr Asn Trp Thr Tyr Va - #l Ser Ala Val His Thr                       220      - #           225      - #           230                  - - GAA GGG AAT TAT GGG GAG AGC GGA ATG GAC GC - #T TTC AAA GAG CTG GCT         1131                                                                       Glu Gly Asn Tyr Gly Glu Ser Gly Met Asp Al - #a Phe Lys Glu Leu Ala                   235          - #       240          - #       245                      - - GCC CAG GAA GGC CTC TGT ATC GCC CAT TCT GA - #C AAA ATC TAC AGC AAC         1179                                                                       Ala Gln Glu Gly Leu Cys Ile Ala His Ser As - #p Lys Ile Tyr Ser Asn               250              - #   255              - #   260                          - - GCT GGG GAG AAG AGC TTT GAC CGA CTC TTG CG - #C AAA CTC CGA GAG AGG         1227                                                                       Ala Gly Glu Lys Ser Phe Asp Arg Leu Leu Ar - #g Lys Leu Arg Glu Arg           265                 2 - #70                 2 - #75                 2 -      #80                                                                              - - CTT CCC AAG GCT AGA GTG GTG GTC TGC TTC TG - #T GAA GGC ATG ACA        GTG     1275                                                                    Leu Pro Lys Ala Arg Val Val Val Cys Phe Cy - #s Glu Gly Met Thr Val                          285  - #               290  - #               295              - - CGA GGA CTC CTG AGC GCC ATG CGG CGC CTT GG - #C GTC GTG GGC GAG TTC         1323                                                                       Arg Gly Leu Leu Ser Ala Met Arg Arg Leu Gl - #y Val Val Gly Glu Phe                       300      - #           305      - #           310                  - - TCA CTC ATT GGA AGT GAT GGA TGG GCA GAC AG - #A GAT GAA GTC ATT GAA         1371                                                                       Ser Leu Ile Gly Ser Asp Gly Trp Ala Asp Ar - #g Asp Glu Val Ile Glu                   315          - #       320          - #       325                      - - GGT TAT GAG GTG GAA GCC AAC GGG GGA ATC AC - #G ATA AAG CTG CAG TCT         1419                                                                       Gly Tyr Glu Val Glu Ala Asn Gly Gly Ile Th - #r Ile Lys Leu Gln Ser               330              - #   335              - #   340                          - - CCA GAG GTC AGG TCA TTT GAT GAT TAT TTC CT - #G AAA CTG AGG CTG GAC         1467                                                                       Pro Glu Val Arg Ser Phe Asp Asp Tyr Phe Le - #u Lys Leu Arg Leu Asp           345                 3 - #50                 3 - #55                 3 -      #60                                                                              - - ACT AAC ACG AGG AAT CCC TGG TTC CCT GAG TT - #C TGG CAA CAT CGG        TTC     1515                                                                    Thr Asn Thr Arg Asn Pro Trp Phe Pro Glu Ph - #e Trp Gln His Arg Phe                          365  - #               370  - #               375              - - CAG TGC CGC CTT CCA GGA CAC CTT CTG GAA AA - #T CCC AAC TTT AAA CGA         1563                                                                       Gln Cys Arg Leu Pro Gly His Leu Leu Glu As - #n Pro Asn Phe Lys Arg                       380      - #           385      - #           390                  - - ATC TGC ACA GGC AAT GAA AGC TTA GAA GAA AA - #C TAT GTC CAG GAC AGT         1611                                                                       Ile Cys Thr Gly Asn Glu Ser Leu Glu Glu As - #n Tyr Val Gln Asp Ser                   395          - #       400          - #       405                      - - AAG ATG GGG TTT GTC ATC AAT GCC ATC TAT GC - #C ATG GCA CAT GGG CTG         1659                                                                       Lys Met Gly Phe Val Ile Asn Ala Ile Tyr Al - #a Met Ala His Gly Leu               410              - #   415              - #   420                          - - CAG AAC ATG CAC CAT GCC CTC TGC CCT GGC CA - #C GTG GGC CTC TGC GAT         1707                                                                       Gln Asn Met His His Ala Leu Cys Pro Gly Hi - #s Val Gly Leu Cys Asp           425                 4 - #30                 4 - #35                 4 -      #40                                                                              - - GCC ATG AAG CCC ATC GAC GGC AGC AAG CTG CT - #G GAC TTC CTC ATC        AAG     1755                                                                    Ala Met Lys Pro Ile Asp Gly Ser Lys Leu Le - #u Asp Phe Leu Ile Lys                          445  - #               450  - #               455              - - TCC TCA TTC ATT GGA GTA TCT GGA GAG GAG GT - #G TGG TTT GAT GAG AAA         1803                                                                       Ser Ser Phe Ile Gly Val Ser Gly Glu Glu Va - #l Trp Phe Asp Glu Lys                       460      - #           465      - #           470                  - - GGA GAC GCT CCT GGA AGG TAT GAT ATC ATG AA - #T CTG CAG TAC ACT GAA         1851                                                                       Gly Asp Ala Pro Gly Arg Tyr Asp Ile Met As - #n Leu Gln Tyr Thr Glu                   475          - #       480          - #       485                      - - GCT AAT CGC TAT GAC TAT GTG CAC GTT GGA AC - #C TGG CAT GAA GGA GTG         1899                                                                       Ala Asn Arg Tyr Asp Tyr Val His Val Gly Th - #r Trp His Glu Gly Val               490              - #   495              - #   500                          - - CTG AAC ATT GAT GAT TAC AAA ATC CAG ATG AA - #C AAG AGT GGA GTG GTG         1947                                                                       Leu Asn Ile Asp Asp Tyr Lys Ile Gln Met As - #n Lys Ser Gly Val Val           505                 5 - #10                 5 - #15                 5 -      #20                                                                              - - CGG TCT GTG TGC AGT GAG CCT TGC TTA AAG GG - #C CAG ATT AAG GTT        ATA     1995                                                                    Arg Ser Val Cys Ser Glu Pro Cys Leu Lys Gl - #y Gln Ile Lys Val Ile                          525  - #               530  - #               535              - - CGG AAA GGA GAA GTG AGC TGC TGC TGG ATT TG - #C GCG GCC TGC AAA GAG         2043                                                                       Arg Lys Gly Glu Val Ser Cys Cys Trp Ile Cy - #s Ala Ala Cys Lys Glu                       540      - #           545      - #           550                  - - AAT GAA TAT GTG CAA GAT GAG TTC ACC TGC AA - #A GCT TGT GAC TTG GGA         2091                                                                       Asn Glu Tyr Val Gln Asp Glu Phe Thr Cys Ly - #s Ala Cys Asp Leu Gly                   555          - #       560          - #       565                      - - TGG TGG CCC AAT GCA GAT CTA ACA GGC TGT GA - #G CCC ATT CCT GTG CGC         2139                                                                       Trp Trp Pro Asn Ala Asp Leu Thr Gly Cys Gl - #u Pro Ile Pro Val Arg               570              - #   575              - #   580                          - - TAT CTT GAG TGG AGC AAC ATC GAA TCC ATT AT - #A GCC ATC GCC TTT TCA         2187                                                                       Tyr Leu Glu Trp Ser Asn Ile Glu Ser Ile Il - #e Ala Ile Ala Phe Ser           585                 5 - #90                 5 - #95                 6 -      #00                                                                              - - TGC CTG GGA ATC CTT GTT ACC TTG TTT GTC AC - #C CTA ATC TTT GTA        CTG     2235                                                                    Cys Leu Gly Ile Leu Val Thr Leu Phe Val Th - #r Leu Ile Phe Val Leu                          605  - #               610  - #               615              - - TAC CGG GAC ACA CCA GTG GTC AAA TCC TCC AG - #T CGG GAG CTC TGC TAC         2283                                                                       Tyr Arg Asp Thr Pro Val Val Lys Ser Ser Se - #r Arg Glu Leu Cys Tyr                       620      - #           625      - #           630                  - - ATC ATC CTA GCT GGC ATC TTC CTT GGT TAT GT - #G TGC CCA TTC ACT CTC         2331                                                                       Ile Ile Leu Ala Gly Ile Phe Leu Gly Tyr Va - #l Cys Pro Phe Thr Leu                   635          - #       640          - #       645                      - - ATT GCC AAA CCT ACT ACC ACC TCC TGC TAC CT - #C CAG CGC CTC TTG GTT         2379                                                                       Ile Ala Lys Pro Thr Thr Thr Ser Cys Tyr Le - #u Gln Arg Leu Leu Val               650              - #   655              - #   660                          - - GGC CTC TCC TCT GCG ATG TGC TAC TCT GCT TT - #A GTG ACT AAA ACC AAT         2427                                                                       Gly Leu Ser Ser Ala Met Cys Tyr Ser Ala Le - #u Val Thr Lys Thr Asn           665                 6 - #70                 6 - #75                 6 -      #80                                                                              - - CGT ATT GCA CGC ATC CTG GCT GGC AGC AAG AA - #G AAG ATC TGC ACC        CGG     2475                                                                    Arg Ile Ala Arg Ile Leu Ala Gly Ser Lys Ly - #s Lys Ile Cys Thr Arg                          685  - #               690  - #               695              - - AAG CCC AGG TTC ATG AGT GCC TGG GCT CAG GT - #G ATC ATT GCC TCA ATT         2523                                                                       Lys Pro Arg Phe Met Ser Ala Trp Ala Gln Va - #l Ile Ile Ala Ser Ile                       700      - #           705      - #           710                  - - CTG ATT AGT GTG CAA CTA ACC CTG GTG GTA AC - #C CTG ATC ATC ATG GAA         2571                                                                       Leu Ile Ser Val Gln Leu Thr Leu Val Val Th - #r Leu Ile Ile Met Glu                   715          - #       720          - #       725                      - - CCC CCT ATG CCC ATT CTG TCC TAC CCA AGT AT - #C AAG GAA GTC TAC CTT         2619                                                                       Pro Pro Met Pro Ile Leu Ser Tyr Pro Ser Il - #e Lys Glu Val Tyr Leu               730              - #   735              - #   740                          - - ATC TGC AAT ACC AGC AAC CTG GGT GTG GTG GC - #C CCT TTG GGC TAC AAT         2667                                                                       Ile Cys Asn Thr Ser Asn Leu Gly Val Val Al - #a Pro Leu Gly Tyr Asn           745                 7 - #50                 7 - #55                 7 -      #60                                                                              - - GGA CTC CTC ATC ATG AGC TGT ACC TAC TAT GC - #C TTC AAG ACC CGC        AAC     2715                                                                    Gly Leu Leu Ile Met Ser Cys Thr Tyr Tyr Al - #a Phe Lys Thr Arg Asn                          765  - #               770  - #               775              - - GTG CCC GCC AAC TTC AAC GAG GCC AAA TAT AT - #C GCG TTC ACC ATG TAC         2763                                                                       Val Pro Ala Asn Phe Asn Glu Ala Lys Tyr Il - #e Ala Phe Thr Met Tyr                       780      - #           785      - #           790                  - - ACC ACC TGT ATC ATC TGG CTA GCT TTT GTG CC - #C ATT TAC TTT GGG AGC         2811                                                                       Thr Thr Cys Ile Ile Trp Leu Ala Phe Val Pr - #o Ile Tyr Phe Gly Ser                   795          - #       800          - #       805                      - - AAC TAC AAG ATC ATC ACA ACT TGC TTT GCA GT - #G AGT CTC AGT GTA ACA         2859                                                                       Asn Tyr Lys Ile Ile Thr Thr Cys Phe Ala Va - #l Ser Leu Ser Val Thr               810              - #   815              - #   820                          - - GTG GCT CTG GGG TGC ATG TTC ACT CCC AAG AT - #G TAC ATC ATT ATT GCC         2907                                                                       Val Ala Leu Gly Cys Met Phe Thr Pro Lys Me - #t Tyr Ile Ile Ile Ala           825                 8 - #30                 8 - #35                 8 -      #40                                                                              - - AAG CCT GAG AGG AAT GTC CGC AGT GCC TTC AC - #C ACC TCT GAT GTT        GTC     2955                                                                    Lys Pro Glu Arg Asn Val Arg Ser Ala Phe Th - #r Thr Ser Asp Val Val                          845  - #               850  - #               855              - - CGC ATG CAT GTT GGC GAT GGC AAG CTG CCC TG - #C CGC TCC AAC ACT TTC         3003                                                                       Arg Met His Val Gly Asp Gly Lys Leu Pro Cy - #s Arg Ser Asn Thr Phe                       860      - #           865      - #           870                  - - CTC AAC ATC TTC CGA AGA AAG AAG GCA GGG GC - #A GGG AAT GCC AAG AAG         3051                                                                       Leu Asn Ile Phe Arg Arg Lys Lys Ala Gly Al - #a Gly Asn Ala Lys Lys                   875          - #       880          - #       885                      - - AGG CAG CCA GAA TTC TCG CCC ACC AGC CAA TG - #T CCG TCG GCA CAT GTG         3099                                                                       Arg Gln Pro Glu Phe Ser Pro Thr Ser Gln Cy - #s Pro Ser Ala His Val               890              - #   895              - #   900                          - - CAG CTT TGAAAACCCC CACACTGCAG TGAATGTTTC TAATGGCAAG TC - #TGTGTCAT          3155                                                                       Gln Leu                                                                       905                                                                            - - GGTCTGAACC AGGTGGAGGA CAGGTGCCCA AGGGACAGCA TATGTGGCAC CG -             #CCTCTCTG   3215                                                                 - - TGCACGTGAA GACCAATGAG ACGGCCTGCA ACCAAACAGC CGTCATCAAA CC -            #CCTCACTA   3275                                                                 - - AAAGTTACCA AGGCTCTGGC AAGAGCCTGA CCTTTTCAGA TACCAG   - #                   3321                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 906 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Val Gly Leu Leu Leu Phe Phe Phe Pro Al - #a Ile Phe Leu Glu Val        1               5 - #                 10 - #                 15              - - Ser Leu Leu Pro Arg Ser Pro Gly Arg Lys Va - #l Leu Leu Ala Gly Ala                   20     - #             25     - #             30                  - - Ser Ser Gln Arg Ser Val Ala Arg Met Asp Gl - #y Asp Val Ile Ile Gly               35         - #         40         - #         45                      - - Ala Leu Phe Ser Val His His Gln Pro Pro Al - #a Glu Lys Val Pro Glu           50             - #     55             - #     60                          - - Arg Lys Cys Gly Glu Ile Arg Glu Gln Tyr Gl - #y Ile Gln Arg Val Glu       65                 - # 70                 - # 75                 - # 80       - - Ala Met Phe His Thr Leu Asp Lys Ile Asn Al - #a Asp Pro Val Leu Leu                       85 - #                 90 - #                 95              - - Pro Asn Ile Thr Leu Gly Ser Glu Ile Arg As - #p Ser Cys Trp His Ser                  100      - #           105      - #           110                  - - Ser Val Ala Leu Glu Gln Ser Ile Glu Phe Il - #e Arg Asp Ser Leu Ile              115          - #       120          - #       125                      - - Ser Ile Arg Asp Glu Lys Asp Gly Ile Asn Ar - #g Cys Leu Pro Asp Gly          130              - #   135              - #   140                          - - Gln Ser Leu Pro Pro Gly Arg Thr Lys Lys Pr - #o Ile Ala Gly Val Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gly Pro Gly Ser Ser Ser Val Ala Ile Gln Va - #l Gln Asn Leu Leu        Gln                                                                                             165  - #               170  - #               175             - - Leu Phe Asp Ile Pro Gln Ile Ala Tyr Ser Al - #a Thr Ser Ile Asp Leu                  180      - #           185      - #           190                  - - Ser Asp Lys Thr Leu Tyr Lys Tyr Phe Leu Ar - #g Val Val Pro Ser Asp              195          - #       200          - #       205                      - - Thr Leu Gln Ala Arg Ala Met Leu Asp Ile Va - #l Lys Arg Tyr Asn Trp          210              - #   215              - #   220                          - - Thr Tyr Val Ser Ala Val His Thr Glu Gly As - #n Tyr Gly Glu Ser Gly      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Met Asp Ala Phe Lys Glu Leu Ala Ala Gln Gl - #u Gly Leu Cys Ile        Ala                                                                                             245  - #               250  - #               255             - - His Ser Asp Lys Ile Tyr Ser Asn Ala Gly Gl - #u Lys Ser Phe Asp Arg                  260      - #           265      - #           270                  - - Leu Leu Arg Lys Leu Arg Glu Arg Leu Pro Ly - #s Ala Arg Val Val Val              275          - #       280          - #       285                      - - Cys Phe Cys Glu Gly Met Thr Val Arg Gly Le - #u Leu Ser Ala Met Arg          290              - #   295              - #   300                          - - Arg Leu Gly Val Val Gly Glu Phe Ser Leu Il - #e Gly Ser Asp Gly Trp      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ala Asp Arg Asp Glu Val Ile Glu Gly Tyr Gl - #u Val Glu Ala Asn        Gly                                                                                             325  - #               330  - #               335             - - Gly Ile Thr Ile Lys Leu Gln Ser Pro Glu Va - #l Arg Ser Phe Asp Asp                  340      - #           345      - #           350                  - - Tyr Phe Leu Lys Leu Arg Leu Asp Thr Asn Th - #r Arg Asn Pro Trp Phe              355          - #       360          - #       365                      - - Pro Glu Phe Trp Gln His Arg Phe Gln Cys Ar - #g Leu Pro Gly His Leu          370              - #   375              - #   380                          - - Leu Glu Asn Pro Asn Phe Lys Arg Ile Cys Th - #r Gly Asn Glu Ser Leu      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Glu Glu Asn Tyr Val Gln Asp Ser Lys Met Gl - #y Phe Val Ile Asn        Ala                                                                                             405  - #               410  - #               415             - - Ile Tyr Ala Met Ala His Gly Leu Gln Asn Me - #t His His Ala Leu Cys                  420      - #           425      - #           430                  - - Pro Gly His Val Gly Leu Cys Asp Ala Met Ly - #s Pro Ile Asp Gly Ser              435          - #       440          - #       445                      - - Lys Leu Leu Asp Phe Leu Ile Lys Ser Ser Ph - #e Ile Gly Val Ser Gly          450              - #   455              - #   460                          - - Glu Glu Val Trp Phe Asp Glu Lys Gly Asp Al - #a Pro Gly Arg Tyr Asp      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Ile Met Asn Leu Gln Tyr Thr Glu Ala Asn Ar - #g Tyr Asp Tyr Val        His                                                                                             485  - #               490  - #               495             - - Val Gly Thr Trp His Glu Gly Val Leu Asn Il - #e Asp Asp Tyr Lys Ile                  500      - #           505      - #           510                  - - Gln Met Asn Lys Ser Gly Val Val Arg Ser Va - #l Cys Ser Glu Pro Cys              515          - #       520          - #       525                      - - Leu Lys Gly Gln Ile Lys Val Ile Arg Lys Gl - #y Glu Val Ser Cys Cys          530              - #   535              - #   540                          - - Trp Ile Cys Ala Ala Cys Lys Glu Asn Glu Ty - #r Val Gln Asp Glu Phe      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Thr Cys Lys Ala Cys Asp Leu Gly Trp Trp Pr - #o Asn Ala Asp Leu        Thr                                                                                             565  - #               570  - #               575             - - Gly Cys Glu Pro Ile Pro Val Arg Tyr Leu Gl - #u Trp Ser Asn Ile Glu                  580      - #           585      - #           590                  - - Ser Ile Ile Ala Ile Ala Phe Ser Cys Leu Gl - #y Ile Leu Val Thr Leu              595          - #       600          - #       605                      - - Phe Val Thr Leu Ile Phe Val Leu Tyr Arg As - #p Thr Pro Val Val Lys          610              - #   615              - #   620                          - - Ser Ser Ser Arg Glu Leu Cys Tyr Ile Ile Le - #u Ala Gly Ile Phe Leu      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Gly Tyr Val Cys Pro Phe Thr Leu Ile Ala Ly - #s Pro Thr Thr Thr        Ser                                                                                             645  - #               650  - #               655             - - Cys Tyr Leu Gln Arg Leu Leu Val Gly Leu Se - #r Ser Ala Met Cys Tyr                  660      - #           665      - #           670                  - - Ser Ala Leu Val Thr Lys Thr Asn Arg Ile Al - #a Arg Ile Leu Ala Gly              675          - #       680          - #       685                      - - Ser Lys Lys Lys Ile Cys Thr Arg Lys Pro Ar - #g Phe Met Ser Ala Trp          690              - #   695              - #   700                          - - Ala Gln Val Ile Ile Ala Ser Ile Leu Ile Se - #r Val Gln Leu Thr Leu      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Val Val Thr Leu Ile Ile Met Glu Pro Pro Me - #t Pro Ile Leu Ser        Tyr                                                                                             725  - #               730  - #               735             - - Pro Ser Ile Lys Glu Val Tyr Leu Ile Cys As - #n Thr Ser Asn Leu Gly                  740      - #           745      - #           750                  - - Val Val Ala Pro Leu Gly Tyr Asn Gly Leu Le - #u Ile Met Ser Cys Thr              755          - #       760          - #       765                      - - Tyr Tyr Ala Phe Lys Thr Arg Asn Val Pro Al - #a Asn Phe Asn Glu Ala          770              - #   775              - #   780                          - - Lys Tyr Ile Ala Phe Thr Met Tyr Thr Thr Cy - #s Ile Ile Trp Leu Ala      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Phe Val Pro Ile Tyr Phe Gly Ser Asn Tyr Ly - #s Ile Ile Thr Thr        Cys                                                                                             805  - #               810  - #               815             - - Phe Ala Val Ser Leu Ser Val Thr Val Ala Le - #u Gly Cys Met Phe Thr                  820      - #           825      - #           830                  - - Pro Lys Met Tyr Ile Ile Ile Ala Lys Pro Gl - #u Arg Asn Val Arg Ser              835          - #       840          - #       845                      - - Ala Phe Thr Thr Ser Asp Val Val Arg Met Hi - #s Val Gly Asp Gly Lys          850              - #   855              - #   860                          - - Leu Pro Cys Arg Ser Asn Thr Phe Leu Asn Il - #e Phe Arg Arg Lys Lys      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - Ala Gly Ala Gly Asn Ala Lys Lys Arg Gln Pr - #o Glu Phe Ser Pro        Thr                                                                                             885  - #               890  - #               895             - - Ser Gln Cys Pro Ser Ala His Val Gln Leu                                              900      - #           905                                         - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 355 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                   - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..354                                                          (D) OTHER INFORMATION: - #/product= "HUMAN MGLUR2 FRAGMENT"          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GCC AAG CCA TCC ACG GCA GTG TGT ACC TTA CG - #G CGT CTT GGT TTG GGC           48                                                                       Ala Lys Pro Ser Thr Ala Val Cys Thr Leu Ar - #g Arg Leu Gly Leu Gly             1               5 - #                 10 - #                 15              - - ACT GCC TTC TCT GTC TGC TAC TCA GCC CTG CT - #C ACC AAG ACC AAC CGC           96                                                                       Thr Ala Phe Ser Val Cys Tyr Ser Ala Leu Le - #u Thr Lys Thr Asn Arg                        20     - #             25     - #             30                  - - ATT GCA CGC ATC TTC GGT GGG GCC CGG GAG GG - #T GCC CAG CGG CCA CGC          144                                                                       Ile Ala Arg Ile Phe Gly Gly Ala Arg Glu Gl - #y Ala Gln Arg Pro Arg                    35         - #         40         - #         45                      - - TTC ATC AGT CCT GCC TCA CAG GTG GCC ATC TG - #C CTG GAA CTT ATC TCG          192                                                                       Phe Ile Ser Pro Ala Ser Gln Val Ala Ile Cy - #s Leu Glu Leu Ile Ser                50             - #     55             - #     60                          - - GGC CAG CTG CTC ATC GTG GTC GCC TGG CTG GT - #G GTG GAG GCA CCG GGC          240                                                                       Gly Gln Leu Leu Ile Val Val Ala Trp Leu Va - #l Val Glu Ala Pro Gly            65                 - # 70                 - # 75                 - # 80       - - ACA GGC AAG GAG ACA GCC CCC GAA CGG CGG GA - #G GTG GTG ACA CTG CGC          288                                                                       Thr Gly Lys Glu Thr Ala Pro Glu Arg Arg Gl - #u Val Val Thr Leu Arg                            85 - #                 90 - #                 95              - - TGC AAC CAC CGC GAT GCA AGT ATG TTG GGC TC - #G CTG GCC TAC AAT GTG          336                                                                       Cys Asn His Arg Asp Ala Ser Met Leu Gly Se - #r Leu Ala Tyr Asn Val                       100      - #           105      - #           110                  - - CTC CTC ATC GCG CTC TGC A        - #                  - #                      - #355                                                                  Leu Leu Ile Ala Leu Cys                                                               115                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 118 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Ala Lys Pro Ser Thr Ala Val Cys Thr Leu Ar - #g Arg Leu Gly Leu Gly        1               5 - #                 10 - #                 15              - - Thr Ala Phe Ser Val Cys Tyr Ser Ala Leu Le - #u Thr Lys Thr Asn Arg                   20     - #             25     - #             30                  - - Ile Ala Arg Ile Phe Gly Gly Ala Arg Glu Gl - #y Ala Gln Arg Pro Arg               35         - #         40         - #         45                      - - Phe Ile Ser Pro Ala Ser Gln Val Ala Ile Cy - #s Leu Glu Leu Ile Ser           50             - #     55             - #     60                          - - Gly Gln Leu Leu Ile Val Val Ala Trp Leu Va - #l Val Glu Ala Pro Gly       65                 - # 70                 - # 75                 - # 80       - - Thr Gly Lys Glu Thr Ala Pro Glu Arg Arg Gl - #u Val Val Thr Leu Arg                       85 - #                 90 - #                 95              - - Cys Asn His Arg Asp Ala Ser Met Leu Gly Se - #r Leu Ala Tyr Asn Val                  100      - #           105      - #           110                  - - Leu Leu Ile Ala Leu Cys                                                          115                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3919 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                   - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1064..3703                                                      (D) OTHER INFORMATION: - #/product= "HUMAN MGLUR3"                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - CGGCCTCCCT GGCTCTCACA CTCCCTCTCT GCTCCCGCTC TCCTAATCTC CT -             #CTGGCATG     60                                                                 - - CGGTCAGCCC CCTGCCCAGG GACCACAGGA GAGTTCTTGT AAGGACTGTT AG -            #TCCCTGCT    120                                                                 - - TACCTGAAAG CCAAGCGCTC TAGCAGAGCT TTAAAGTTGG AGCCGCCACC CT -            #CCCTACCG    180                                                                 - - CCCCATGCCC CTTCACCCCA CTCCGAAATT CACCGACCTT TGCATGCACT GC -            #CTAAGGAT    240                                                                 - - TTCAGAGTGA GGCAAAGCAG TCGGCAAATC TACCCTGGCT TTTCGTATAA AA -            #ATCCTCTC    300                                                                 - - GTCTAGGTAC CCTGGCTCAC TGAAGACTCT GCAGATATAC CCTTATAAGA GG -            #GAGGGTGG    360                                                                 - - GGGAGGGAAA AGAACGAGAG AGGGAGGAAA GAATGAAAAG GAGAGGATGC CA -            #GGAGGTCC    420                                                                 - - GTGCTTCTGC CAAGAGTCCC AATTAGATGC GACGGCTTCA GCCTGGTCAA GG -            #TGAAGGAA    480                                                                 - - AGTTGCTTCC GCGCCTAGGA AGTGGGTTTG CCTGATAAGA GAAGGAGGAG GG -            #GACTCGGC    540                                                                 - - TGGGAAGAGC TCCCCTCCCC TCCGCGGAAG ACCACTGGGT CCCCTCTTTC GG -            #CAACCTCC    600                                                                 - - TCCCTCTCTT CTACTCCACC CCTCCGTTTT CCCACTCCCC ACTGACTCGG AT -            #GCCTGGAT    660                                                                 - - GTTCTGCCAC CGGGCAGTGG TCCAGCGTGC AGCCGGGAGG GGGCAGGGGC AG -            #GGGGCACT    720                                                                 - - GTGACAGGAA GCTGCGCGCA CAAGTTGGCC ATTTCGAGGG CAAAATAAGT TC -            #TCCCTTGG    780                                                                 - - ATTTGGAAAG GACAAAGCCA GTAAGCTACC TCTTTTGTGT CGGATGAGGA GG -            #ACCAACCA    840                                                                 - - TGAGCCAGAG CCCGGGTGCA GGCTCACCGC CGCCGCTGCC ACCGCGGTCA GC -            #TCCAGTTC    900                                                                 - - CTGCCAGGAG TTGTCGGTGC GAGGAATTTT GTGACAGGCT CTGTTAGTCT GT -            #TCCTCCCT    960                                                                 - - TATTTGAAGG ACAGGCCAAA GATCCAGTTT GGAAATGAGA GAGGACTAGC AT -            #GACACATT   1020                                                                 - - GGCTCCACCA TTGATATCTC CCAGAGGTAC AGAAACAGGA TTC ATG AA - #G ATG        TTG     1075                                                                                      - #                  - #            Met Lys Met Le -      #u                                                                                                - #                  - #              1                      - - ACA AGA CTG CAA GTT CTT ACC TTA GCT TTG TT - #T TCA AAG GGA TTT        TTA     1123                                                                    Thr Arg Leu Gln Val Leu Thr Leu Ala Leu Ph - #e Ser Lys Gly Phe Leu            5                - #  10                - #  15                - #  20       - - CTC TCT TTA GGG GAC CAT AAC TTT CTA AGG AG - #A GAG ATT AAA ATA GAA         1171                                                                       Leu Ser Leu Gly Asp His Asn Phe Leu Arg Ar - #g Glu Ile Lys Ile Glu                            25 - #                 30 - #                 35              - - GGT GAC CTT GTT TTA GGG GGC CTG TTT CCT AT - #T AAC GAA AAA GGC ACT         1219                                                                       Gly Asp Leu Val Leu Gly Gly Leu Phe Pro Il - #e Asn Glu Lys Gly Thr                        40     - #             45     - #             50                  - - GGA ACT GAA GAA TGT GGG CGA ATC AAT GAA GA - #C CGA GGG ATT CAA CGC         1267                                                                       Gly Thr Glu Glu Cys Gly Arg Ile Asn Glu As - #p Arg Gly Ile Gln Arg                    55         - #         60         - #         65                      - - CTG GAA GCC ATG TTG TTT GCT ATT GAT GAA AT - #C AAC AAA GAT GAT TAC         1315                                                                       Leu Glu Ala Met Leu Phe Ala Ile Asp Glu Il - #e Asn Lys Asp Asp Tyr                70             - #     75             - #     80                          - - TTG CTA CCA GGA GTG AAG TTG GGT GTT CAC AT - #T TTG GAT ACA TGT TCA         1363                                                                       Leu Leu Pro Gly Val Lys Leu Gly Val His Il - #e Leu Asp Thr Cys Ser            85                 - # 90                 - # 95                 - #100       - - AGG GAT ACC TAT GCA TTG GAG CAA TCA CTG GA - #G TTT GTC AGG GCA TCT         1411                                                                       Arg Asp Thr Tyr Ala Leu Glu Gln Ser Leu Gl - #u Phe Val Arg Ala Ser                           105  - #               110  - #               115              - - TTG ACA AAA GTG GAT GAA GCT GAG TAT ATG TG - #T CCT GAT GGA TCC TAT         1459                                                                       Leu Thr Lys Val Asp Glu Ala Glu Tyr Met Cy - #s Pro Asp Gly Ser Tyr                       120      - #           125      - #           130                  - - GCC ATT CAA GAA AAC ATC CCA CTT CTC ATT GC - #A GGG GTC ATT GGT GGC         1507                                                                       Ala Ile Gln Glu Asn Ile Pro Leu Leu Ile Al - #a Gly Val Ile Gly Gly                   135          - #       140          - #       145                      - - TCT TAT AGC AGT GTT TCC ATA CAG GTG GCA AA - #C CTG CTG CGG CTC TTC         1555                                                                       Ser Tyr Ser Ser Val Ser Ile Gln Val Ala As - #n Leu Leu Arg Leu Phe               150              - #   155              - #   160                          - - CAG ATC CCT CAG ATC AGC TAC GCA TCC ACC AG - #C GCC AAA CTC AGT GAT         1603                                                                       Gln Ile Pro Gln Ile Ser Tyr Ala Ser Thr Se - #r Ala Lys Leu Ser Asp           165                 1 - #70                 1 - #75                 1 -      #80                                                                              - - AAG TCG CGC TAT GAT TAC TTT GCC AGG ACC GT - #G CCC CCC GAC TTC        TAC     1651                                                                    Lys Ser Arg Tyr Asp Tyr Phe Ala Arg Thr Va - #l Pro Pro Asp Phe Tyr                          185  - #               190  - #               195              - - CAG GCC AAA GCC ATG GCT GAG ATC TTG CGC TT - #C TTC AAC TGG ACC TAC         1699                                                                       Gln Ala Lys Ala Met Ala Glu Ile Leu Arg Ph - #e Phe Asn Trp Thr Tyr                       200      - #           205      - #           210                  - - GTG TCC ACA GTA GCC TCC GAG GGT GAT TAC GG - #G GAG ACA GGG ATC GAG         1747                                                                       Val Ser Thr Val Ala Ser Glu Gly Asp Tyr Gl - #y Glu Thr Gly Ile Glu                   215          - #       220          - #       225                      - - GCC TTC GAG CAG GAA GCC CGC CTG CGC AAC AT - #C TGC ATC GCT ACG GCG         1795                                                                       Ala Phe Glu Gln Glu Ala Arg Leu Arg Asn Il - #e Cys Ile Ala Thr Ala               230              - #   235              - #   240                          - - GAG AAG GTG GGC CGC TCC AAC ATC CGC AAG TC - #C TAC GAC AGC GTG ATC         1843                                                                       Glu Lys Val Gly Arg Ser Asn Ile Arg Lys Se - #r Tyr Asp Ser Val Ile           245                 2 - #50                 2 - #55                 2 -      #60                                                                              - - CGA GAA CTG TTG CAG AAG CCC AAC GCG CGC GT - #C GTG GTC CTC TTC        ATG     1891                                                                    Arg Glu Leu Leu Gln Lys Pro Asn Ala Arg Va - #l Val Val Leu Phe Met                          265  - #               270  - #               275              - - CGC AGC GAC GAC TCG CGG GAG CTC ATT GCA GC - #C GCC AGC CGC GCC AAT         1939                                                                       Arg Ser Asp Asp Ser Arg Glu Leu Ile Ala Al - #a Ala Ser Arg Ala Asn                       280      - #           285      - #           290                  - - GCC TCC TTC ACC TGG GTG GCC AGC GAC GGT TG - #G GGC GCG CAG GAG AGC         1987                                                                       Ala Ser Phe Thr Trp Val Ala Ser Asp Gly Tr - #p Gly Ala Gln Glu Ser                   295          - #       300          - #       305                      - - ATC ATC AAG GGC AGC GAG CAT GTG GCC TAC GG - #C GAC ATC ACC CTG GAG         2035                                                                       Ile Ile Lys Gly Ser Glu His Val Ala Tyr Gl - #y Asp Ile Thr Leu Glu               310              - #   315              - #   320                          - - CTG GCC TCC CAG CCT GTC CGC CAG TTC GGC CG - #C TAC TTC CAG AGC CTC         2083                                                                       Leu Ala Ser Gln Pro Val Arg Gln Phe Gly Ar - #g Tyr Phe Gln Ser Leu           325                 3 - #30                 3 - #35                 3 -      #40                                                                              - - AAC CCC TAC AAC AAC CAC CGC AAC CCC TGG TT - #C CGG GAC TTC TGG        GAG     2131                                                                    Asn Pro Tyr Asn Asn His Arg Asn Pro Trp Ph - #e Arg Asp Phe Trp Glu                          345  - #               350  - #               355              - - CAA AAG TTT CAG TGC AGC CTC CAG AAC AAA CG - #C AAC CAC AGG CGC GTC         2179                                                                       Gln Lys Phe Gln Cys Ser Leu Gln Asn Lys Ar - #g Asn His Arg Arg Val                       360      - #           365      - #           370                  - - TGC GAA AAG CAC CTG GCC ATC GAC AGC AGC AA - #C TAC GAG CAA GAG TCC         2227                                                                       Cys Glu Lys His Leu Ala Ile Asp Ser Ser As - #n Tyr Glu Gln Glu Ser                   375          - #       380          - #       385                      - - AAG ATC ATG TTT GTG GTG AAC GCG GTG TAT GC - #C ATG GCC CAC GCT TTG         2275                                                                       Lys Ile Met Phe Val Val Asn Ala Val Tyr Al - #a Met Ala His Ala Leu               390              - #   395              - #   400                          - - CAC AAA ATG CAG CGC ACC CTC TGT CCC AAC AC - #T ACC AAG CTT TGT GAT         2323                                                                       His Lys Met Gln Arg Thr Leu Cys Pro Asn Th - #r Thr Lys Leu Cys Asp           405                 4 - #10                 4 - #15                 4 -      #20                                                                              - - GCT ATG AAG ATC CTG GAT GGG AAG AAG TTG TA - #C AAG GAT TAC TTG        CTG     2371                                                                    Ala Met Lys Ile Leu Asp Gly Lys Lys Leu Ty - #r Lys Asp Tyr Leu Leu                          425  - #               430  - #               435              - - AAA ATC AAC TTC ACG GCT CCA TTC AAC CCA AA - #T AAA GAT GCA GAT AGC         2419                                                                       Lys Ile Asn Phe Thr Ala Pro Phe Asn Pro As - #n Lys Asp Ala Asp Ser                       440      - #           445      - #           450                  - - ATA GTC AAG TTT GAC ACT TTT GGA GAT GGA AT - #G GGG CGA TAC AAC GTG         2467                                                                       Ile Val Lys Phe Asp Thr Phe Gly Asp Gly Me - #t Gly Arg Tyr Asn Val                   455          - #       460          - #       465                      - - TTC AAT TTC CAA AAT GTA GGT GGG AAG TAT TC - #C TAC TTG AAA GTT GGT         2515                                                                       Phe Asn Phe Gln Asn Val Gly Gly Lys Tyr Se - #r Tyr Leu Lys Val Gly               470              - #   475              - #   480                          - - CAC TGG GCA GAA ACC TTA TCG CTA GAT GTC AA - #C TCT ATC CAC TGG TCC         2563                                                                       His Trp Ala Glu Thr Leu Ser Leu Asp Val As - #n Ser Ile His Trp Ser           485                 4 - #90                 4 - #95                 5 -      #00                                                                              - - CGG AAC TCA GTC CCC ACT TCC CAG TGC AGC GA - #C CCC TGT GCC CCC        AAT     2611                                                                    Arg Asn Ser Val Pro Thr Ser Gln Cys Ser As - #p Pro Cys Ala Pro Asn                          505  - #               510  - #               515              - - GAA ATG AAG AAT ATG CAA CCA GGG GAT GTC TG - #C TGC TGG ATT TGC ATC         2659                                                                       Glu Met Lys Asn Met Gln Pro Gly Asp Val Cy - #s Cys Trp Ile Cys Ile                       520      - #           525      - #           530                  - - CCC TGT GAA CCC TAC GAA TAC CTG GCT GAT GA - #G TTT ACC TGT ATG GAT         2707                                                                       Pro Cys Glu Pro Tyr Glu Tyr Leu Ala Asp Gl - #u Phe Thr Cys Met Asp                   535          - #       540          - #       545                      - - TGT GGG TCT GGA CAG TGG CCC ACT GCA GAC CT - #A ACT GGA TGC TAT GAC         2755                                                                       Cys Gly Ser Gly Gln Trp Pro Thr Ala Asp Le - #u Thr Gly Cys Tyr Asp               550              - #   555              - #   560                          - - CTT CCT GAG GAC TAC ATC AGG TGG GAA GAC GC - #C TGG GCC ATT GGC CCA         2803                                                                       Leu Pro Glu Asp Tyr Ile Arg Trp Glu Asp Al - #a Trp Ala Ile Gly Pro           565                 5 - #70                 5 - #75                 5 -      #80                                                                              - - GTC ACC ATT GCC TGT CTG GGT TTT ATG TGT AC - #A TGC ATG GTT GTA        ACT     2851                                                                    Val Thr Ile Ala Cys Leu Gly Phe Met Cys Th - #r Cys Met Val Val Thr                          585  - #               590  - #               595              - - GTT TTT ATC AAG CAC AAC AAC ACA CCC TTG GT - #C AAA GCA TCG GGC CGA         2899                                                                       Val Phe Ile Lys His Asn Asn Thr Pro Leu Va - #l Lys Ala Ser Gly Arg                       600      - #           605      - #           610                  - - GAA CTC TGC TAC ATC TTA TTG TTT GGG GTT GG - #C CTG TCA TAC TGC ATG         2947                                                                       Glu Leu Cys Tyr Ile Leu Leu Phe Gly Val Gl - #y Leu Ser Tyr Cys Met                   615          - #       620          - #       625                      - - ACA TTC TTC TTC ATT GCC AAG CCA TCA CCA GT - #C ATC TGT GCA TTG CGC         2995                                                                       Thr Phe Phe Phe Ile Ala Lys Pro Ser Pro Va - #l Ile Cys Ala Leu Arg               630              - #   635              - #   640                          - - CGA CTC GGG CTG GGG AGT TCC TTC GCT ATC TG - #T TAC TCA GCC CTG CTG         3043                                                                       Arg Leu Gly Leu Gly Ser Ser Phe Ala Ile Cy - #s Tyr Ser Ala Leu Leu           645                 6 - #50                 6 - #55                 6 -      #60                                                                              - - ACC AAG ACA AAC TGC ATT GCC CGC ATC TTC GA - #T GGG GTC AAG AAT        GGC     3091                                                                    Thr Lys Thr Asn Cys Ile Ala Arg Ile Phe As - #p Gly Val Lys Asn Gly                          665  - #               670  - #               675              - - GCT CAG AGG CCA AAA TTC ATC AGC CCC AGT TC - #T CAG GTT TTC ATC TGC         3139                                                                       Ala Gln Arg Pro Lys Phe Ile Ser Pro Ser Se - #r Gln Val Phe Ile Cys                       680      - #           685      - #           690                  - - CTG GGT CTG ATC CTG GTG CAA ATT GTG ATG GT - #G TCT GTG TGG CTC ATC         3187                                                                       Leu Gly Leu Ile Leu Val Gln Ile Val Met Va - #l Ser Val Trp Leu Ile                   695          - #       700          - #       705                      - - CTG GAG GCC CCA GGC ACC AGG AGG TAT ACC CT - #T GCA GAG AAG CGG GAA         3235                                                                       Leu Glu Ala Pro Gly Thr Arg Arg Tyr Thr Le - #u Ala Glu Lys Arg Glu               710              - #   715              - #   720                          - - ACA GTC ATC CTA AAA TGC AAT GTC AAA GAT TC - #C AGC ATG TTG ATC TCT         3283                                                                       Thr Val Ile Leu Lys Cys Asn Val Lys Asp Se - #r Ser Met Leu Ile Ser           725                 7 - #30                 7 - #35                 7 -      #40                                                                              - - CTT ACC TAC GAT GTG ATC CTG GTG ATC TTA TG - #C ACT GTG TAC GCC        TTC     3331                                                                    Leu Thr Tyr Asp Val Ile Leu Val Ile Leu Cy - #s Thr Val Tyr Ala Phe                          745  - #               750  - #               755              - - AAA ACG CGG AAG TGC CCA GAA AAT TTC AAC GA - #A GCT AAG TTC ATA GGT         3379                                                                       Lys Thr Arg Lys Cys Pro Glu Asn Phe Asn Gl - #u Ala Lys Phe Ile Gly                       760      - #           765      - #           770                  - - TTT ACC ATG TAC ACC ACG TGC ATC ATC TGG TT - #G GCC TTC CTC CCT ATA         3427                                                                       Phe Thr Met Tyr Thr Thr Cys Ile Ile Trp Le - #u Ala Phe Leu Pro Ile                   775          - #       780          - #       785                      - - TTT TAT GTG ACA TCA AGT GAC TAC AGA GTG CA - #G ACG ACA ACC ATG TGC         3475                                                                       Phe Tyr Val Thr Ser Ser Asp Tyr Arg Val Gl - #n Thr Thr Thr Met Cys               790              - #   795              - #   800                          - - ATC TCT GTC AGC CTG AGT GGC TTT GTG GTC TT - #G GGC TGT TTG TTT GCA         3523                                                                       Ile Ser Val Ser Leu Ser Gly Phe Val Val Le - #u Gly Cys Leu Phe Ala           805                 8 - #10                 8 - #15                 8 -      #20                                                                              - - CCC AAG GTT CAC ATC ATC CTG TTT CAA CCC CA - #G AAG AAT GTT GTC        ACA     3571                                                                    Pro Lys Val His Ile Ile Leu Phe Gln Pro Gl - #n Lys Asn Val Val Thr                          825  - #               830  - #               835              - - CAC AGA CTG CAC CTC AAC AGG TTC AGT GTC AG - #T GGA ACT GGG ACC ACA         3619                                                                       His Arg Leu His Leu Asn Arg Phe Ser Val Se - #r Gly Thr Gly Thr Thr                       840      - #           845      - #           850                  - - TAC TCT CAG TCC TCT GCA AGC ACG TAT GTG CC - #A ACG GTG TGC AAT GGG         3667                                                                       Tyr Ser Gln Ser Ser Ala Ser Thr Tyr Val Pr - #o Thr Val Cys Asn Gly                   855          - #       860          - #       865                      - - CGG GAA GTC CTC GAC TCC ACC ACC TCA TCT CT - #G TGATTGTGAA TTGCAGTTC    A   3720                                                                       Arg Glu Val Leu Asp Ser Thr Thr Ser Ser Le - #u                                   870              - #   875              - #   880                          - - GTTCTTGTGT TTTTAGACTG TTAGACAAAA GTGCTCACGT GCAGCTCCAG AA -             #TATGGAAA   3780                                                                 - - CAGAGCAAAA GAACAACCCT AGTACCTTTT TTTAGAAACA GTACGATAAA TT -            #ATTTTTGA   3840                                                                 - - GGACTGTATA TAGTGATGTG CTAGAACTTT CTAGGCTGAG TCTAGTGCCC CT -            #ATTATTAA   3900                                                                 - - CAGTCCGAGT GTACGTACC             - #                  - #                     391 - #9                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 879 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Lys Met Leu Thr Arg Leu Gln Val Leu Th - #r Leu Ala Leu Phe Ser        1               5 - #                 10 - #                 15              - - Lys Gly Phe Leu Leu Ser Leu Gly Asp His As - #n Phe Leu Arg Arg Glu                   20     - #             25     - #             30                  - - Ile Lys Ile Glu Gly Asp Leu Val Leu Gly Gl - #y Leu Phe Pro Ile Asn               35         - #         40         - #         45                      - - Glu Lys Gly Thr Gly Thr Glu Glu Cys Gly Ar - #g Ile Asn Glu Asp Arg           50             - #     55             - #     60                          - - Gly Ile Gln Arg Leu Glu Ala Met Leu Phe Al - #a Ile Asp Glu Ile Asn       65                 - # 70                 - # 75                 - # 80       - - Lys Asp Asp Tyr Leu Leu Pro Gly Val Lys Le - #u Gly Val His Ile Leu                       85 - #                 90 - #                 95              - - Asp Thr Cys Ser Arg Asp Thr Tyr Ala Leu Gl - #u Gln Ser Leu Glu Phe                  100      - #           105      - #           110                  - - Val Arg Ala Ser Leu Thr Lys Val Asp Glu Al - #a Glu Tyr Met Cys Pro              115          - #       120          - #       125                      - - Asp Gly Ser Tyr Ala Ile Gln Glu Asn Ile Pr - #o Leu Leu Ile Ala Gly          130              - #   135              - #   140                          - - Val Ile Gly Gly Ser Tyr Ser Ser Val Ser Il - #e Gln Val Ala Asn Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Arg Leu Phe Gln Ile Pro Gln Ile Ser Ty - #r Ala Ser Thr Ser        Ala                                                                                             165  - #               170  - #               175             - - Lys Leu Ser Asp Lys Ser Arg Tyr Asp Tyr Ph - #e Ala Arg Thr Val Pro                  180      - #           185      - #           190                  - - Pro Asp Phe Tyr Gln Ala Lys Ala Met Ala Gl - #u Ile Leu Arg Phe Phe              195          - #       200          - #       205                      - - Asn Trp Thr Tyr Val Ser Thr Val Ala Ser Gl - #u Gly Asp Tyr Gly Glu          210              - #   215              - #   220                          - - Thr Gly Ile Glu Ala Phe Glu Gln Glu Ala Ar - #g Leu Arg Asn Ile Cys      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ile Ala Thr Ala Glu Lys Val Gly Arg Ser As - #n Ile Arg Lys Ser        Tyr                                                                                             245  - #               250  - #               255             - - Asp Ser Val Ile Arg Glu Leu Leu Gln Lys Pr - #o Asn Ala Arg Val Val                  260      - #           265      - #           270                  - - Val Leu Phe Met Arg Ser Asp Asp Ser Arg Gl - #u Leu Ile Ala Ala Ala              275          - #       280          - #       285                      - - Ser Arg Ala Asn Ala Ser Phe Thr Trp Val Al - #a Ser Asp Gly Trp Gly          290              - #   295              - #   300                          - - Ala Gln Glu Ser Ile Ile Lys Gly Ser Glu Hi - #s Val Ala Tyr Gly Asp      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ile Thr Leu Glu Leu Ala Ser Gln Pro Val Ar - #g Gln Phe Gly Arg        Tyr                                                                                             325  - #               330  - #               335             - - Phe Gln Ser Leu Asn Pro Tyr Asn Asn His Ar - #g Asn Pro Trp Phe Arg                  340      - #           345      - #           350                  - - Asp Phe Trp Glu Gln Lys Phe Gln Cys Ser Le - #u Gln Asn Lys Arg Asn              355          - #       360          - #       365                      - - His Arg Arg Val Cys Glu Lys His Leu Ala Il - #e Asp Ser Ser Asn Tyr          370              - #   375              - #   380                          - - Glu Gln Glu Ser Lys Ile Met Phe Val Val As - #n Ala Val Tyr Ala Met      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ala His Ala Leu His Lys Met Gln Arg Thr Le - #u Cys Pro Asn Thr        Thr                                                                                             405  - #               410  - #               415             - - Lys Leu Cys Asp Ala Met Lys Ile Leu Asp Gl - #y Lys Lys Leu Tyr Lys                  420      - #           425      - #           430                  - - Asp Tyr Leu Leu Lys Ile Asn Phe Thr Ala Pr - #o Phe Asn Pro Asn Lys              435          - #       440          - #       445                      - - Asp Ala Asp Ser Ile Val Lys Phe Asp Thr Ph - #e Gly Asp Gly Met Gly          450              - #   455              - #   460                          - - Arg Tyr Asn Val Phe Asn Phe Gln Asn Val Gl - #y Gly Lys Tyr Ser Tyr      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Leu Lys Val Gly His Trp Ala Glu Thr Leu Se - #r Leu Asp Val Asn        Ser                                                                                             485  - #               490  - #               495             - - Ile His Trp Ser Arg Asn Ser Val Pro Thr Se - #r Gln Cys Ser Asp Pro                  500      - #           505      - #           510                  - - Cys Ala Pro Asn Glu Met Lys Asn Met Gln Pr - #o Gly Asp Val Cys Cys              515          - #       520          - #       525                      - - Trp Ile Cys Ile Pro Cys Glu Pro Tyr Glu Ty - #r Leu Ala Asp Glu Phe          530              - #   535              - #   540                          - - Thr Cys Met Asp Cys Gly Ser Gly Gln Trp Pr - #o Thr Ala Asp Leu Thr      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Gly Cys Tyr Asp Leu Pro Glu Asp Tyr Ile Ar - #g Trp Glu Asp Ala        Trp                                                                                             565  - #               570  - #               575             - - Ala Ile Gly Pro Val Thr Ile Ala Cys Leu Gl - #y Phe Met Cys Thr Cys                  580      - #           585      - #           590                  - - Met Val Val Thr Val Phe Ile Lys His Asn As - #n Thr Pro Leu Val Lys              595          - #       600          - #       605                      - - Ala Ser Gly Arg Glu Leu Cys Tyr Ile Leu Le - #u Phe Gly Val Gly Leu          610              - #   615              - #   620                          - - Ser Tyr Cys Met Thr Phe Phe Phe Ile Ala Ly - #s Pro Ser Pro Val Ile      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Cys Ala Leu Arg Arg Leu Gly Leu Gly Ser Se - #r Phe Ala Ile Cys        Tyr                                                                                             645  - #               650  - #               655             - - Ser Ala Leu Leu Thr Lys Thr Asn Cys Ile Al - #a Arg Ile Phe Asp Gly                  660      - #           665      - #           670                  - - Val Lys Asn Gly Ala Gln Arg Pro Lys Phe Il - #e Ser Pro Ser Ser Gln              675          - #       680          - #       685                      - - Val Phe Ile Cys Leu Gly Leu Ile Leu Val Gl - #n Ile Val Met Val Ser          690              - #   695              - #   700                          - - Val Trp Leu Ile Leu Glu Ala Pro Gly Thr Ar - #g Arg Tyr Thr Leu Ala      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Glu Lys Arg Glu Thr Val Ile Leu Lys Cys As - #n Val Lys Asp Ser        Ser                                                                                             725  - #               730  - #               735             - - Met Leu Ile Ser Leu Thr Tyr Asp Val Ile Le - #u Val Ile Leu Cys Thr                  740      - #           745      - #           750                  - - Val Tyr Ala Phe Lys Thr Arg Lys Cys Pro Gl - #u Asn Phe Asn Glu Ala              755          - #       760          - #       765                      - - Lys Phe Ile Gly Phe Thr Met Tyr Thr Thr Cy - #s Ile Ile Trp Leu Ala          770              - #   775              - #   780                          - - Phe Leu Pro Ile Phe Tyr Val Thr Ser Ser As - #p Tyr Arg Val Gln Thr      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Thr Thr Met Cys Ile Ser Val Ser Leu Ser Gl - #y Phe Val Val Leu        Gly                                                                                             805  - #               810  - #               815             - - Cys Leu Phe Ala Pro Lys Val His Ile Ile Le - #u Phe Gln Pro Gln Lys                  820      - #           825      - #           830                  - - Asn Val Val Thr His Arg Leu His Leu Asn Ar - #g Phe Ser Val Ser Gly              835          - #       840          - #       845                      - - Thr Gly Thr Thr Tyr Ser Gln Ser Ser Ala Se - #r Thr Tyr Val Pro Thr          850              - #   855              - #   860                          - - Val Cys Asn Gly Arg Glu Val Leu Asp Ser Th - #r Thr Ser Ser Leu          865                 8 - #70                 8 - #75                            - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4085 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                   - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 370..3912                                                       (D) OTHER INFORMATION: - #/product= "HUMAN MGLUR5A"                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - CAGCTCGGCT GTTCTGCGCA CGCTGAGCGG AGGGAATGAG CTTGAGATCA TC -             #TTGGGGGG     60                                                                 - - GAAGCCGGGG ACTGGAGAGG CCGGCTCTGC CCTGCTGATC CCCGTGGCCC AA -            #CTTTTCGG    120                                                                 - - GGGGCTAGCT AGACCGAGTC TCACTGCTCG CAGCGCAGCC AACAGGGGGG TT -            #TAGAAGAT    180                                                                 - - CATGACCACA TGGATCATCT AACTAAATGG TACATGGGGA CAAAATGGTC CT -            #TTAGAAAA    240                                                                 - - TACATCTGAA TTGCTGGCTA ATTTCTTGAT TTGCGACTCA ACGTAGGACA TC -            #GCTTGTTC    300                                                                 - - GTAGCTATCA GAACCCTCCT GAATTTTCCC CACCATGCTA TCTTTATTGG CT -            #TGAACTCC    360                                                                 - - TTTCCTAAA ATG GTC CTT CTG TTG ATC CTG TCA GTC - # TTA CTT TGG AAA            408                                                                                Met Val Leu Leu Leu - #Ile Leu Ser Val Leu Leu Trp Lys                          1      - #         5         - #         10                        - - GAA GAT GTC CGT GGG AGT GCA CAG TCC AGT GA - #G AGG AGG GTG GTG GCT          456                                                                       Glu Asp Val Arg Gly Ser Ala Gln Ser Ser Gl - #u Arg Arg Val Val Ala                15             - #     20             - #     25                          - - CAC ATG CCG GGT GAC ATC ATT ATT GGA GCT CT - #C TTT TCT GTT CAT CAC          504                                                                       His Met Pro Gly Asp Ile Ile Ile Gly Ala Le - #u Phe Ser Val His His            30                 - # 35                 - # 40                 - # 45       - - CAG CCT ACT GTG GAC AAA GTT CAT GAG AGG AA - #G TGT GGG GCG GTC CGT          552                                                                       Gln Pro Thr Val Asp Lys Val His Glu Arg Ly - #s Cys Gly Ala Val Arg                            50 - #                 55 - #                 60              - - GAA CAG TAT GGC ATT CAG AGA GTG GAG GCC AT - #G CTG CAT ACC CTG GAA          600                                                                       Glu Gln Tyr Gly Ile Gln Arg Val Glu Ala Me - #t Leu His Thr Leu Glu                        65     - #             70     - #             75                  - - AGG ATC AAT TCA GAC CCC ACA CTC TTG CCC AA - #C ATC ACA CTG GGC TGT          648                                                                       Arg Ile Asn Ser Asp Pro Thr Leu Leu Pro As - #n Ile Thr Leu Gly Cys                    80         - #         85         - #         90                      - - GAG ATA AGG GAC TCC TGC TGG CAT TCG GCT GT - #G GCC CTA GAG CAG AGC          696                                                                       Glu Ile Arg Asp Ser Cys Trp His Ser Ala Va - #l Ala Leu Glu Gln Ser                95             - #    100             - #    105                          - - ATT GAG TTC ATA AGA GAT TCC CTC ATT TCT TC - #A GAA GAG GAA GAA GGC          744                                                                       Ile Glu Phe Ile Arg Asp Ser Leu Ile Ser Se - #r Glu Glu Glu Glu Gly           110                 1 - #15                 1 - #20                 1 -      #25                                                                              - - TTG GTA CGC TGT GTG GAT GGC TCC TCC TCT TC - #C TTC CGC TCC AAG        AAG      792                                                                    Leu Val Arg Cys Val Asp Gly Ser Ser Ser Se - #r Phe Arg Ser Lys Lys                          130  - #               135  - #               140              - - CCC ATA GTA GGG GTC ATT GGG CCT GGC TCC AG - #T TCT GTA GCC ATT CAG          840                                                                       Pro Ile Val Gly Val Ile Gly Pro Gly Ser Se - #r Ser Val Ala Ile Gln                       145      - #           150      - #           155                  - - GTC CAG AAT TTG CTC CAG CTT TTC AAC ATA CC - #T CAG ATT GCT TAC TCA          888                                                                       Val Gln Asn Leu Leu Gln Leu Phe Asn Ile Pr - #o Gln Ile Ala Tyr Ser                   160          - #       165          - #       170                      - - GCA ACC AGC ATG GAT CTG AGT GAC AAG ACT CT - #G TTC AAA TAT TTC ATG          936                                                                       Ala Thr Ser Met Asp Leu Ser Asp Lys Thr Le - #u Phe Lys Tyr Phe Met               175              - #   180              - #   185                          - - AGG GTT GTG CCT TCA GAT GCT CAG CAG GCA AG - #G GCC ATG GTG GAC ATA          984                                                                       Arg Val Val Pro Ser Asp Ala Gln Gln Ala Ar - #g Ala Met Val Asp Ile           190                 1 - #95                 2 - #00                 2 -      #05                                                                              - - GTG AAG AGG TAC AAC TGG ACC TAT GTA TCA GC - #C GTG CAC ACA GAA        GGC     1032                                                                    Val Lys Arg Tyr Asn Trp Thr Tyr Val Ser Al - #a Val His Thr Glu Gly                          210  - #               215  - #               220              - - AAC TAT GGA GAA AGT GGG ATG GAA GCC TCC AA - #A GAT ATG TCA GCG AAG         1080                                                                       Asn Tyr Gly Glu Ser Gly Met Glu Ala Ser Ly - #s Asp Met Ser Ala Lys                       225      - #           230      - #           235                  - - GAA GGG ATT TGC ATC GCC CAC TCT TAC AAA AT - #C TAC AGT AAT GCA GGG         1128                                                                       Glu Gly Ile Cys Ile Ala His Ser Tyr Lys Il - #e Tyr Ser Asn Ala Gly                   240          - #       245          - #       250                      - - GAG CAG AGC TTT GAT AAG CTG CTG AAG AAG CT - #C ACA AGT CAC TTG CCC         1176                                                                       Glu Gln Ser Phe Asp Lys Leu Leu Lys Lys Le - #u Thr Ser His Leu Pro               255              - #   260              - #   265                          - - AAG GCC CGG GTG GTG GCC TGC TTC TGT GAG GG - #C ATG ACG GTG AGA GGT         1224                                                                       Lys Ala Arg Val Val Ala Cys Phe Cys Glu Gl - #y Met Thr Val Arg Gly           270                 2 - #75                 2 - #80                 2 -      #85                                                                              - - CTG CTG ATG GCC ATG AGG CGC CTG GGT CTA GC - #G GGA GAA TTT CTG        CTT     1272                                                                    Leu Leu Met Ala Met Arg Arg Leu Gly Leu Al - #a Gly Glu Phe Leu Leu                          290  - #               295  - #               300              - - CTG GGC AGT GAT GGC TGG GCT GAC AGG TAT GA - #T GTG ACA GAT GGA TAT         1320                                                                       Leu Gly Ser Asp Gly Trp Ala Asp Arg Tyr As - #p Val Thr Asp Gly Tyr                       305      - #           310      - #           315                  - - CAG CGA GAA GCT GTT GGT GGC ATC ACA ATC AA - #G CTC CAA TCT CCC GAT         1368                                                                       Gln Arg Glu Ala Val Gly Gly Ile Thr Ile Ly - #s Leu Gln Ser Pro Asp                   320          - #       325          - #       330                      - - GTC AAG TGG TTT GAT GAT TAT TAT CTG AAG CT - #C CGG CCA GAA ACA AAC         1416                                                                       Val Lys Trp Phe Asp Asp Tyr Tyr Leu Lys Le - #u Arg Pro Glu Thr Asn               335              - #   340              - #   345                          - - CAC CGA AAC CCT TGG TTT CAA GAA TTT TGG CA - #G CAT CGT TTT CAG TGC         1464                                                                       His Arg Asn Pro Trp Phe Gln Glu Phe Trp Gl - #n His Arg Phe Gln Cys           350                 3 - #55                 3 - #60                 3 -      #65                                                                              - - CGA CTG GAA GCG TTT CCA CAG GAG AAC AGC AA - #A TAC AAC AAG ACT        TGC     1512                                                                    Arg Leu Glu Ala Phe Pro Gln Glu Asn Ser Ly - #s Tyr Asn Lys Thr Cys                          370  - #               375  - #               380              - - AAT AGT TCT CTG ACT CTG AAA ACA CAT CAT GT - #T CAG GAT TCC AAA ATG         1560                                                                       Asn Ser Ser Leu Thr Leu Lys Thr His His Va - #l Gln Asp Ser Lys Met                       385      - #           390      - #           395                  - - GGA TTT GTG ATC AAC GCC ATC TAT TCG ATG GC - #C TAT GGG CTC CAC AAC         1608                                                                       Gly Phe Val Ile Asn Ala Ile Tyr Ser Met Al - #a Tyr Gly Leu His Asn                   400          - #       405          - #       410                      - - ATG CAG ATG TCC CTC TGC CCA GGC TAT GCA GG - #A CTC TGT GAT GCC ATG         1656                                                                       Met Gln Met Ser Leu Cys Pro Gly Tyr Ala Gl - #y Leu Cys Asp Ala Met               415              - #   420              - #   425                          - - AAG CCA ATT GAT GGA CGG AAA CTT TTG GAG TC - #C CTG ATG AAA ACC AAT         1704                                                                       Lys Pro Ile Asp Gly Arg Lys Leu Leu Glu Se - #r Leu Met Lys Thr Asn           430                 4 - #35                 4 - #40                 4 -      #45                                                                              - - TTT ACT GGG GTT TCT GGA GAT ACG ATC CTA TT - #C GAT GAG AAT GGA        GAC     1752                                                                    Phe Thr Gly Val Ser Gly Asp Thr Ile Leu Ph - #e Asp Glu Asn Gly Asp                          450  - #               455  - #               460              - - TCT CCA GGA AGG TAT GAA ATA ATG AAT TTC AA - #G GAA ATG GGA AAA GAT         1800                                                                       Ser Pro Gly Arg Tyr Glu Ile Met Asn Phe Ly - #s Glu Met Gly Lys Asp                       465      - #           470      - #           475                  - - TAC TTT GAT TAT ATC AAC GTT GGA AGT TGG GA - #C AAT GGA GAA TTA AAA         1848                                                                       Tyr Phe Asp Tyr Ile Asn Val Gly Ser Trp As - #p Asn Gly Glu Leu Lys                   480          - #       485          - #       490                      - - ATG GAT GAT GAT GAA GTA TGG TCC AAG AAA AG - #C AAC ATC ATC AGA TCT         1896                                                                       Met Asp Asp Asp Glu Val Trp Ser Lys Lys Se - #r Asn Ile Ile Arg Ser               495              - #   500              - #   505                          - - GTG TGC AGT GAA CCA TGT GAG AAA GGC CAG AT - #C AAG GTG ATC CGA AAG         1944                                                                       Val Cys Ser Glu Pro Cys Glu Lys Gly Gln Il - #e Lys Val Ile Arg Lys           510                 5 - #15                 5 - #20                 5 -      #25                                                                              - - GGA GAA GTC AGC TGT TGT TGG ACC TGT ACA CC - #T TGT AAG GAG AAT        GAG     1992                                                                    Gly Glu Val Ser Cys Cys Trp Thr Cys Thr Pr - #o Cys Lys Glu Asn Glu                          530  - #               535  - #               540              - - TAT GTC TTT GAT GAG TAC ACA TGC AAG GCA TG - #C CAA CTG GGG TCT TGG         2040                                                                       Tyr Val Phe Asp Glu Tyr Thr Cys Lys Ala Cy - #s Gln Leu Gly Ser Trp                       545      - #           550      - #           555                  - - CCC ACT GAT GAT CTC ACA GGT TGT GAC TTG AT - #C CCA GTA CAG TAT CTT         2088                                                                       Pro Thr Asp Asp Leu Thr Gly Cys Asp Leu Il - #e Pro Val Gln Tyr Leu                   560          - #       565          - #       570                      - - CGA TGG GGT GAC CCT GAA CCC ATT GCA GCT GT - #G GTG TTT GCC TGC CTT         2136                                                                       Arg Trp Gly Asp Pro Glu Pro Ile Ala Ala Va - #l Val Phe Ala Cys Leu               575              - #   580              - #   585                          - - GGC CTC CTG GCC ACC CTG TTT GTT ACT GTA GT - #C TTC ATC ATT TAC CGT         2184                                                                       Gly Leu Leu Ala Thr Leu Phe Val Thr Val Va - #l Phe Ile Ile Tyr Arg           590                 5 - #95                 6 - #00                 6 -      #05                                                                              - - GAT ACA CCA GTA GTC AAG TCC TCA AGC AGG GA - #A CTC TGC TAC ATT        ATC     2232                                                                    Asp Thr Pro Val Val Lys Ser Ser Ser Arg Gl - #u Leu Cys Tyr Ile Ile                          610  - #               615  - #               620              - - CTT GCT GGC ATC TGC CTG GGC TAC TTA TGT AC - #C TTC TGC CTC ATT GCG         2280                                                                       Leu Ala Gly Ile Cys Leu Gly Tyr Leu Cys Th - #r Phe Cys Leu Ile Ala                       625      - #           630      - #           635                  - - AAG CCC AAA CAG ATT TAC TGC TAC CTT CAG AG - #A ATT GGC ATT GGT CTC         2328                                                                       Lys Pro Lys Gln Ile Tyr Cys Tyr Leu Gln Ar - #g Ile Gly Ile Gly Leu                   640          - #       645          - #       650                      - - TCC CCA GCC ATG AGC TAC TCA GCC CTT GTA AC - #A AAG ACC AAC CGT ATT         2376                                                                       Ser Pro Ala Met Ser Tyr Ser Ala Leu Val Th - #r Lys Thr Asn Arg Ile               655              - #   660              - #   665                          - - GCA AGG ATC CTG GCT GGC AGC AAG AAG AAG AT - #C TGT ACC CCC AAG CCC         2424                                                                       Ala Arg Ile Leu Ala Gly Ser Lys Lys Lys Il - #e Cys Thr Pro Lys Pro           670                 6 - #75                 6 - #80                 6 -      #85                                                                              - - AGA TTC ATG AGT GCC TGT GCC CAG CTA GTG AT - #T GCT TTC ATT CTC        ATA     2472                                                                    Arg Phe Met Ser Ala Cys Ala Gln Leu Val Il - #e Ala Phe Ile Leu Ile                          690  - #               695  - #               700              - - TGC ATC CAG TTG GGC ATC ATC GTT GCC CTC TT - #T ATA ATG GAG CCT CCT         2520                                                                       Cys Ile Gln Leu Gly Ile Ile Val Ala Leu Ph - #e Ile Met Glu Pro Pro                       705      - #           710      - #           715                  - - GAC ATA ATG CAT GAC TAC CCA AGC ATT CGA GA - #A GTC TAC CTG ATC TGT         2568                                                                       Asp Ile Met His Asp Tyr Pro Ser Ile Arg Gl - #u Val Tyr Leu Ile Cys                   720          - #       725          - #       730                      - - AAC ACC ACC AAC CTA GGA GTT GTC ACT CCA CT - #T GGA AAC AAT GGA TTG         2616                                                                       Asn Thr Thr Asn Leu Gly Val Val Thr Pro Le - #u Gly Asn Asn Gly Leu               735              - #   740              - #   745                          - - TTG ATT TTG AGC TGC ACC TTC TAT GCG TTC AA - #G ACC AGA AAT GTT CCA         2664                                                                       Leu Ile Leu Ser Cys Thr Phe Tyr Ala Phe Ly - #s Thr Arg Asn Val Pro           750                 7 - #55                 7 - #60                 7 -      #65                                                                              - - GCT AAC TTC CCC GAG GCC AAG TAT ATC GCC TT - #C ACA ATG TAC ACG        ACC     2712                                                                    Ala Asn Phe Pro Glu Ala Lys Tyr Ile Ala Ph - #e Thr Met Tyr Thr Thr                          770  - #               775  - #               780              - - TGC ATT ATA TGG CTA GCT TTT GTT CCA ATC TA - #C TTT GGC AGC AAC TAC         2760                                                                       Cys Ile Ile Trp Leu Ala Phe Val Pro Ile Ty - #r Phe Gly Ser Asn Tyr                       785      - #           790      - #           795                  - - AAA ATC ATC ACC ATG TGT TTC TCG GTC AGC CT - #C AGT GCC ACA GTG GCC         2808                                                                       Lys Ile Ile Thr Met Cys Phe Ser Val Ser Le - #u Ser Ala Thr Val Ala                   800          - #       805          - #       810                      - - CTA GGC TGC ATG TTT GTG CCG AAG GTG TAC AT - #C ATC CTG GCC AAA CCA         2856                                                                       Leu Gly Cys Met Phe Val Pro Lys Val Tyr Il - #e Ile Leu Ala Lys Pro               815              - #   820              - #   825                          - - GAG AGA AAC GTG CGC AGC GCC TTC ACC ACA TC - #T ACC GTG GTG CGC ATG         2904                                                                       Glu Arg Asn Val Arg Ser Ala Phe Thr Thr Se - #r Thr Val Val Arg Met           830                 8 - #35                 8 - #40                 8 -      #45                                                                              - - CAT GTA GGG GAT GGC AAG TCA TCC TCC GCA GC - #C AGC AGA TCC AGC        AGC     2952                                                                    His Val Gly Asp Gly Lys Ser Ser Ser Ala Al - #a Ser Arg Ser Ser Ser                          850  - #               855  - #               860              - - CTA GTC AAC CTG TGG AAG AGA AGG GGC TCC TC - #T GGG GAA ACC TTA AGT         3000                                                                       Leu Val Asn Leu Trp Lys Arg Arg Gly Ser Se - #r Gly Glu Thr Leu Ser                       865      - #           870      - #           875                  - - TCC AAT GGA AAA TCC GTC ACG TGG GCC CAG AA - #T GAG AAG AGC AGC CGG         3048                                                                       Ser Asn Gly Lys Ser Val Thr Trp Ala Gln As - #n Glu Lys Ser Ser Arg                   880          - #       885          - #       890                      - - GGG CAG CAC CTG TGG CAG CGC CTG TCC ATC CA - #C ATC AAC AAG AAA GAA         3096                                                                       Gly Gln His Leu Trp Gln Arg Leu Ser Ile Hi - #s Ile Asn Lys Lys Glu               895              - #   900              - #   905                          - - AAC CCC AAC CAA ACG GCC GTC ATC AAG CCC TT - #C CCC AAG AGC ACG GAG         3144                                                                       Asn Pro Asn Gln Thr Ala Val Ile Lys Pro Ph - #e Pro Lys Ser Thr Glu           910                 9 - #15                 9 - #20                 9 -      #25                                                                              - - AGC CGT GGC CTG GGC GCT GGC GCT GGC GCA GG - #C GGG AGC GCT GGG        GGC     3192                                                                    Ser Arg Gly Leu Gly Ala Gly Ala Gly Ala Gl - #y Gly Ser Ala Gly Gly                          930  - #               935  - #               940              - - GTG GGG GCC ACG GGC GGT GCG GGC TGC GCA GG - #C GCC GGC CCA GGC GGG         3240                                                                       Val Gly Ala Thr Gly Gly Ala Gly Cys Ala Gl - #y Ala Gly Pro Gly Gly                       945      - #           950      - #           955                  - - CCC GAG TCC CCA GAC GCC GGC CCC AAG GCG CT - #G TAT GAT GTG GCC GAG         3288                                                                       Pro Glu Ser Pro Asp Ala Gly Pro Lys Ala Le - #u Tyr Asp Val Ala Glu                   960          - #       965          - #       970                      - - GCT GAG GAG CAC TTC CCG GCG CCC GCG CGG CC - #G CGC TCA CCG TCG CCC         3336                                                                       Ala Glu Glu His Phe Pro Ala Pro Ala Arg Pr - #o Arg Ser Pro Ser Pro               975              - #   980              - #   985                          - - ATC AGC ACG CTG AGC CAC CGC GCG GGC TCG GC - #C AGC CGC ACG GAC GAC         3384                                                                       Ile Ser Thr Leu Ser His Arg Ala Gly Ser Al - #a Ser Arg Thr Asp Asp           990                 9 - #95                 1 - #000                1005       - - GAT GTG CCG TCG CTG CAC TCG GAG CCT GTG GC - #G CGC AGC AGC TCC TCG         3432                                                                       Asp Val Pro Ser Leu His Ser Glu Pro Val Al - #a Arg Ser Ser Ser Ser                           1010 - #               1015  - #              1020             - - CAG GGC TCC CTC ATG GAG CAG ATC AGC AGT GT - #G GTC ACC CGC TTC ACG         3480                                                                       Gln Gly Ser Leu Met Glu Gln Ile Ser Ser Va - #l Val Thr Arg Phe Thr                       1025     - #           1030      - #          1035                 - - GCC AAC ATC AGC GAG CTC AAC TCC ATG ATG CT - #G TCC ACC GCG GCC CCC         3528                                                                       Ala Asn Ile Ser Glu Leu Asn Ser Met Met Le - #u Ser Thr Ala Ala Pro                   1040         - #       1045          - #      1050                     - - AGC CCC GGC GTC GGC GCC CCG CTC TGC TCG TC - #C TAC CTG ATC CCC AAA         3576                                                                       Ser Pro Gly Val Gly Ala Pro Leu Cys Ser Se - #r Tyr Leu Ile Pro Lys               1055             - #   1060              - #  1065                         - - GAG ATC CAG TTG CCC ACG ACC ATG ACG ACC TT - #T GCC GAA ATC CAG CCT         3624                                                                       Glu Ile Gln Leu Pro Thr Thr Met Thr Thr Ph - #e Ala Glu Ile Gln Pro           1070                1075 - #                1080 - #               1085        - - CTG CCG GCC ATC GAA GTC ACG GGC GGC GCT CA - #G CCC GCG GCA GGG GCG         3672                                                                       Leu Pro Ala Ile Glu Val Thr Gly Gly Ala Gl - #n Pro Ala Ala Gly Ala                           1090 - #               1095  - #              1100             - - CAG GCG GCT GGG GAC GCG GCC CGG GAG AGC CC - #C GCG GCC GGT CCC GAG         3720                                                                       Gln Ala Ala Gly Asp Ala Ala Arg Glu Ser Pr - #o Ala Ala Gly Pro Glu                       1105     - #           1110      - #          1115                 - - GCT GCG GCC GCC AAG CCA GAC CTG GAG GAG CT - #G GTG GCT CTC ACC CCG         3768                                                                       Ala Ala Ala Ala Lys Pro Asp Leu Glu Glu Le - #u Val Ala Leu Thr Pro                   1120         - #       1125          - #      1130                     - - CCG TCC CCC TTC AGA GAC TCG GTG GAC TCG GG - #G AGC ACA ACC CCC AAC         3816                                                                       Pro Ser Pro Phe Arg Asp Ser Val Asp Ser Gl - #y Ser Thr Thr Pro Asn               1135             - #   1140              - #  1145                         - - TCG CCA GTG TCC GAG TCG GCC CTC TGT ATC CC - #G TCG TCT CCC AAA TAT         3864                                                                       Ser Pro Val Ser Glu Ser Ala Leu Cys Ile Pr - #o Ser Ser Pro Lys Tyr           1150                1155 - #                1160 - #               1165        - - GAC ACT CTT ATC ATA AGA GAT TAC ACT CAG AG - #C TCC TCG TCG TTG         TGAATGTC3919                                                                    Asp Thr Leu Ile Ile Arg Asp Tyr Thr Gln Se - #r Ser Ser Ser Leu                              1170 - #               1175  - #              1180             - - TGGAAAGCAC GCCGGCCTGC GCGTGCGGAG CGGAGCCCCC CGTGTTCACA CA -             #CACACAAT   3979                                                                 - - GGCAAGCATA GTCGCCTGGT TACGGCCCAG GGGGAAGATG CCAAGGGCAC CC -            #CTTAATGG   4039                                                                 - - AAACACGAGA TCAGTAGTGC TATCTCATGA CAACCGACGA AGAAAC   - #                   4085                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1180 amino - #acids                                               (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Met Val Leu Leu Leu Ile Leu Ser Val Leu Le - #u Trp Lys Glu Asp Val        1               5 - #                 10 - #                 15              - - Arg Gly Ser Ala Gln Ser Ser Glu Arg Arg Va - #l Val Ala His Met Pro                   20     - #             25     - #             30                  - - Gly Asp Ile Ile Ile Gly Ala Leu Phe Ser Va - #l His His Gln Pro Thr               35         - #         40         - #         45                      - - Val Asp Lys Val His Glu Arg Lys Cys Gly Al - #a Val Arg Glu Gln Tyr           50             - #     55             - #     60                          - - Gly Ile Gln Arg Val Glu Ala Met Leu His Th - #r Leu Glu Arg Ile Asn       65                 - # 70                 - # 75                 - # 80       - - Ser Asp Pro Thr Leu Leu Pro Asn Ile Thr Le - #u Gly Cys Glu Ile Arg                       85 - #                 90 - #                 95              - - Asp Ser Cys Trp His Ser Ala Val Ala Leu Gl - #u Gln Ser Ile Glu Phe                  100      - #           105      - #           110                  - - Ile Arg Asp Ser Leu Ile Ser Ser Glu Glu Gl - #u Glu Gly Leu Val Arg              115          - #       120          - #       125                      - - Cys Val Asp Gly Ser Ser Ser Ser Phe Arg Se - #r Lys Lys Pro Ile Val          130              - #   135              - #   140                          - - Gly Val Ile Gly Pro Gly Ser Ser Ser Val Al - #a Ile Gln Val Gln Asn      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Leu Gln Leu Phe Asn Ile Pro Gln Ile Al - #a Tyr Ser Ala Thr        Ser                                                                                             165  - #               170  - #               175             - - Met Asp Leu Ser Asp Lys Thr Leu Phe Lys Ty - #r Phe Met Arg Val Val                  180      - #           185      - #           190                  - - Pro Ser Asp Ala Gln Gln Ala Arg Ala Met Va - #l Asp Ile Val Lys Arg              195          - #       200          - #       205                      - - Tyr Asn Trp Thr Tyr Val Ser Ala Val His Th - #r Glu Gly Asn Tyr Gly          210              - #   215              - #   220                          - - Glu Ser Gly Met Glu Ala Ser Lys Asp Met Se - #r Ala Lys Glu Gly Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Cys Ile Ala His Ser Tyr Lys Ile Tyr Ser As - #n Ala Gly Glu Gln        Ser                                                                                             245  - #               250  - #               255             - - Phe Asp Lys Leu Leu Lys Lys Leu Thr Ser Hi - #s Leu Pro Lys Ala Arg                  260      - #           265      - #           270                  - - Val Val Ala Cys Phe Cys Glu Gly Met Thr Va - #l Arg Gly Leu Leu Met              275          - #       280          - #       285                      - - Ala Met Arg Arg Leu Gly Leu Ala Gly Glu Ph - #e Leu Leu Leu Gly Ser          290              - #   295              - #   300                          - - Asp Gly Trp Ala Asp Arg Tyr Asp Val Thr As - #p Gly Tyr Gln Arg Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ala Val Gly Gly Ile Thr Ile Lys Leu Gln Se - #r Pro Asp Val Lys        Trp                                                                                             325  - #               330  - #               335             - - Phe Asp Asp Tyr Tyr Leu Lys Leu Arg Pro Gl - #u Thr Asn His Arg Asn                  340      - #           345      - #           350                  - - Pro Trp Phe Gln Glu Phe Trp Gln His Arg Ph - #e Gln Cys Arg Leu Glu              355          - #       360          - #       365                      - - Ala Phe Pro Gln Glu Asn Ser Lys Tyr Asn Ly - #s Thr Cys Asn Ser Ser          370              - #   375              - #   380                          - - Leu Thr Leu Lys Thr His His Val Gln Asp Se - #r Lys Met Gly Phe Val      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ile Asn Ala Ile Tyr Ser Met Ala Tyr Gly Le - #u His Asn Met Gln        Met                                                                                             405  - #               410  - #               415             - - Ser Leu Cys Pro Gly Tyr Ala Gly Leu Cys As - #p Ala Met Lys Pro Ile                  420      - #           425      - #           430                  - - Asp Gly Arg Lys Leu Leu Glu Ser Leu Met Ly - #s Thr Asn Phe Thr Gly              435          - #       440          - #       445                      - - Val Ser Gly Asp Thr Ile Leu Phe Asp Glu As - #n Gly Asp Ser Pro Gly          450              - #   455              - #   460                          - - Arg Tyr Glu Ile Met Asn Phe Lys Glu Met Gl - #y Lys Asp Tyr Phe Asp      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Tyr Ile Asn Val Gly Ser Trp Asp Asn Gly Gl - #u Leu Lys Met Asp        Asp                                                                                             485  - #               490  - #               495             - - Asp Glu Val Trp Ser Lys Lys Ser Asn Ile Il - #e Arg Ser Val Cys Ser                  500      - #           505      - #           510                  - - Glu Pro Cys Glu Lys Gly Gln Ile Lys Val Il - #e Arg Lys Gly Glu Val              515          - #       520          - #       525                      - - Ser Cys Cys Trp Thr Cys Thr Pro Cys Lys Gl - #u Asn Glu Tyr Val Phe          530              - #   535              - #   540                          - - Asp Glu Tyr Thr Cys Lys Ala Cys Gln Leu Gl - #y Ser Trp Pro Thr Asp      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Asp Leu Thr Gly Cys Asp Leu Ile Pro Val Gl - #n Tyr Leu Arg Trp        Gly                                                                                             565  - #               570  - #               575             - - Asp Pro Glu Pro Ile Ala Ala Val Val Phe Al - #a Cys Leu Gly Leu Leu                  580      - #           585      - #           590                  - - Ala Thr Leu Phe Val Thr Val Val Phe Ile Il - #e Tyr Arg Asp Thr Pro              595          - #       600          - #       605                      - - Val Val Lys Ser Ser Ser Arg Glu Leu Cys Ty - #r Ile Ile Leu Ala Gly          610              - #   615              - #   620                          - - Ile Cys Leu Gly Tyr Leu Cys Thr Phe Cys Le - #u Ile Ala Lys Pro Lys      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Gln Ile Tyr Cys Tyr Leu Gln Arg Ile Gly Il - #e Gly Leu Ser Pro        Ala                                                                                             645  - #               650  - #               655             - - Met Ser Tyr Ser Ala Leu Val Thr Lys Thr As - #n Arg Ile Ala Arg Ile                  660      - #           665      - #           670                  - - Leu Ala Gly Ser Lys Lys Lys Ile Cys Thr Pr - #o Lys Pro Arg Phe Met              675          - #       680          - #       685                      - - Ser Ala Cys Ala Gln Leu Val Ile Ala Phe Il - #e Leu Ile Cys Ile Gln          690              - #   695              - #   700                          - - Leu Gly Ile Ile Val Ala Leu Phe Ile Met Gl - #u Pro Pro Asp Ile Met      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - His Asp Tyr Pro Ser Ile Arg Glu Val Tyr Le - #u Ile Cys Asn Thr        Thr                                                                                             725  - #               730  - #               735             - - Asn Leu Gly Val Val Thr Pro Leu Gly Asn As - #n Gly Leu Leu Ile Leu                  740      - #           745      - #           750                  - - Ser Cys Thr Phe Tyr Ala Phe Lys Thr Arg As - #n Val Pro Ala Asn Phe              755          - #       760          - #       765                      - - Pro Glu Ala Lys Tyr Ile Ala Phe Thr Met Ty - #r Thr Thr Cys Ile Ile          770              - #   775              - #   780                          - - Trp Leu Ala Phe Val Pro Ile Tyr Phe Gly Se - #r Asn Tyr Lys Ile Ile      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Thr Met Cys Phe Ser Val Ser Leu Ser Ala Th - #r Val Ala Leu Gly        Cys                                                                                             805  - #               810  - #               815             - - Met Phe Val Pro Lys Val Tyr Ile Ile Leu Al - #a Lys Pro Glu Arg Asn                  820      - #           825      - #           830                  - - Val Arg Ser Ala Phe Thr Thr Ser Thr Val Va - #l Arg Met His Val Gly              835          - #       840          - #       845                      - - Asp Gly Lys Ser Ser Ser Ala Ala Ser Arg Se - #r Ser Ser Leu Val Asn          850              - #   855              - #   860                          - - Leu Trp Lys Arg Arg Gly Ser Ser Gly Glu Th - #r Leu Ser Ser Asn Gly      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - Lys Ser Val Thr Trp Ala Gln Asn Glu Lys Se - #r Ser Arg Gly Gln        His                                                                                             885  - #               890  - #               895             - - Leu Trp Gln Arg Leu Ser Ile His Ile Asn Ly - #s Lys Glu Asn Pro Asn                  900      - #           905      - #           910                  - - Gln Thr Ala Val Ile Lys Pro Phe Pro Lys Se - #r Thr Glu Ser Arg Gly              915          - #       920          - #       925                      - - Leu Gly Ala Gly Ala Gly Ala Gly Gly Ser Al - #a Gly Gly Val Gly Ala          930              - #   935              - #   940                          - - Thr Gly Gly Ala Gly Cys Ala Gly Ala Gly Pr - #o Gly Gly Pro Glu Ser      945                 9 - #50                 9 - #55                 9 -      #60                                                                              - - Pro Asp Ala Gly Pro Lys Ala Leu Tyr Asp Va - #l Ala Glu Ala Glu        Glu                                                                                             965  - #               970  - #               975             - - His Phe Pro Ala Pro Ala Arg Pro Arg Ser Pr - #o Ser Pro Ile Ser Thr                  980      - #           985      - #           990                  - - Leu Ser His Arg Ala Gly Ser Ala Ser Arg Th - #r Asp Asp Asp Val Pro              995          - #       1000          - #      1005                     - - Ser Leu His Ser Glu Pro Val Ala Arg Ser Se - #r Ser Ser Gln Gly Ser          1010             - #   1015              - #  1020                         - - Leu Met Glu Gln Ile Ser Ser Val Val Thr Ar - #g Phe Thr Ala Asn Ile      1025                1030 - #                1035 - #               1040        - - Ser Glu Leu Asn Ser Met Met Leu Ser Thr Al - #a Ala Pro Ser Pro Gly                      1045 - #               1050  - #              1055             - - Val Gly Ala Pro Leu Cys Ser Ser Tyr Leu Il - #e Pro Lys Glu Ile Gln                  1060     - #           1065      - #          1070                 - - Leu Pro Thr Thr Met Thr Thr Phe Ala Glu Il - #e Gln Pro Leu Pro Ala              1075         - #       1080          - #      1085                     - - Ile Glu Val Thr Gly Gly Ala Gln Pro Ala Al - #a Gly Ala Gln Ala Ala          1090             - #   1095              - #  1100                         - - Gly Asp Ala Ala Arg Glu Ser Pro Ala Ala Gl - #y Pro Glu Ala Ala Ala      1105                1110 - #                1115 - #               1120        - - Ala Lys Pro Asp Leu Glu Glu Leu Val Ala Le - #u Thr Pro Pro Ser Pro                      1125 - #               1130  - #              1135             - - Phe Arg Asp Ser Val Asp Ser Gly Ser Thr Th - #r Pro Asn Ser Pro Val                  1140     - #           1145      - #          1150                 - - Ser Glu Ser Ala Leu Cys Ile Pro Ser Ser Pr - #o Lys Tyr Asp Thr Leu              1155         - #       1160          - #      1165                     - - Ile Ile Arg Asp Tyr Thr Gln Ser Ser Ser Se - #r Leu                          1170             - #   1175              - #  1180                         - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4181 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                   - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 370..4008                                                       (D) OTHER INFORMATION: - #/product= "HUMAN MGLUR5B"                                /note= - #"Variant of MGLUR5A with 96 base pair                               insertion - #between nucleotides 2998 and 2999."                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - CAGCTCGGCT GTTCTGCGCA CGCTGAGCGG AGGGAATGAG CTTGAGATCA TC -             #TTGGGGGG     60                                                                 - - GAAGCCGGGG ACTGGAGAGG CCGGCTCTGC CCTGCTGATC CCCGTGGCCC AA -            #CTTTTCGG    120                                                                 - - GGGGCTAGCT AGACCGAGTC TCACTGCTCG CAGCGCAGCC AACAGGGGGG TT -            #TAGAAGAT    180                                                                 - - CATGACCACA TGGATCATCT AACTAAATGG TACATGGGGA CAAAATGGTC CT -            #TTAGAAAA    240                                                                 - - TACATCTGAA TTGCTGGCTA ATTTCTTGAT TTGCGACTCA ACGTAGGACA TC -            #GCTTGTTC    300                                                                 - - GTAGCTATCA GAACCCTCCT GAATTTTCCC CACCATGCTA TCTTTATTGG CT -            #TGAACTCC    360                                                                 - - TTTCCTAAA ATG GTC CTT CTG TTG ATC CTG TCA GTC - # TTA CTT TGG AAA            408                                                                                Met Val Leu Leu Leu - #Ile Leu Ser Val Leu Leu Trp Lys                          1      - #         5         - #         10                        - - GAA GAT GTC CGT GGG AGT GCA CAG TCC AGT GA - #G AGG AGG GTG GTG GCT          456                                                                       Glu Asp Val Arg Gly Ser Ala Gln Ser Ser Gl - #u Arg Arg Val Val Ala                15             - #     20             - #     25                          - - CAC ATG CCG GGT GAC ATC ATT ATT GGA GCT CT - #C TTT TCT GTT CAT CAC          504                                                                       His Met Pro Gly Asp Ile Ile Ile Gly Ala Le - #u Phe Ser Val His His            30                 - # 35                 - # 40                 - # 45       - - CAG CCT ACT GTG GAC AAA GTT CAT GAG AGG AA - #G TGT GGG GCG GTC CGT          552                                                                       Gln Pro Thr Val Asp Lys Val His Glu Arg Ly - #s Cys Gly Ala Val Arg                            50 - #                 55 - #                 60              - - GAA CAG TAT GGC ATT CAG AGA GTG GAG GCC AT - #G CTG CAT ACC CTG GAA          600                                                                       Glu Gln Tyr Gly Ile Gln Arg Val Glu Ala Me - #t Leu His Thr Leu Glu                        65     - #             70     - #             75                  - - AGG ATC AAT TCA GAC CCC ACA CTC TTG CCC AA - #C ATC ACA CTG GGC TGT          648                                                                       Arg Ile Asn Ser Asp Pro Thr Leu Leu Pro As - #n Ile Thr Leu Gly Cys                    80         - #         85         - #         90                      - - GAG ATA AGG GAC TCC TGC TGG CAT TCG GCT GT - #G GCC CTA GAG CAG AGC          696                                                                       Glu Ile Arg Asp Ser Cys Trp His Ser Ala Va - #l Ala Leu Glu Gln Ser                95             - #    100             - #    105                          - - ATT GAG TTC ATA AGA GAT TCC CTC ATT TCT TC - #A GAA GAG GAA GAA GGC          744                                                                       Ile Glu Phe Ile Arg Asp Ser Leu Ile Ser Se - #r Glu Glu Glu Glu Gly           110                 1 - #15                 1 - #20                 1 -      #25                                                                              - - TTG GTA CGC TGT GTG GAT GGC TCC TCC TCT TC - #C TTC CGC TCC AAG        AAG      792                                                                    Leu Val Arg Cys Val Asp Gly Ser Ser Ser Se - #r Phe Arg Ser Lys Lys                          130  - #               135  - #               140              - - CCC ATA GTA GGG GTC ATT GGG CCT GGC TCC AG - #T TCT GTA GCC ATT CAG          840                                                                       Pro Ile Val Gly Val Ile Gly Pro Gly Ser Se - #r Ser Val Ala Ile Gln                       145      - #           150      - #           155                  - - GTC CAG AAT TTG CTC CAG CTT TTC AAC ATA CC - #T CAG ATT GCT TAC TCA          888                                                                       Val Gln Asn Leu Leu Gln Leu Phe Asn Ile Pr - #o Gln Ile Ala Tyr Ser                   160          - #       165          - #       170                      - - GCA ACC AGC ATG GAT CTG AGT GAC AAG ACT CT - #G TTC AAA TAT TTC ATG          936                                                                       Ala Thr Ser Met Asp Leu Ser Asp Lys Thr Le - #u Phe Lys Tyr Phe Met               175              - #   180              - #   185                          - - AGG GTT GTG CCT TCA GAT GCT CAG CAG GCA AG - #G GCC ATG GTG GAC ATA          984                                                                       Arg Val Val Pro Ser Asp Ala Gln Gln Ala Ar - #g Ala Met Val Asp Ile           190                 1 - #95                 2 - #00                 2 -      #05                                                                              - - GTG AAG AGG TAC AAC TGG ACC TAT GTA TCA GC - #C GTG CAC ACA GAA        GGC     1032                                                                    Val Lys Arg Tyr Asn Trp Thr Tyr Val Ser Al - #a Val His Thr Glu Gly                          210  - #               215  - #               220              - - AAC TAT GGA GAA AGT GGG ATG GAA GCC TCC AA - #A GAT ATG TCA GCG AAG         1080                                                                       Asn Tyr Gly Glu Ser Gly Met Glu Ala Ser Ly - #s Asp Met Ser Ala Lys                       225      - #           230      - #           235                  - - GAA GGG ATT TGC ATC GCC CAC TCT TAC AAA AT - #C TAC AGT AAT GCA GGG         1128                                                                       Glu Gly Ile Cys Ile Ala His Ser Tyr Lys Il - #e Tyr Ser Asn Ala Gly                   240          - #       245          - #       250                      - - GAG CAG AGC TTT GAT AAG CTG CTG AAG AAG CT - #C ACA AGT CAC TTG CCC         1176                                                                       Glu Gln Ser Phe Asp Lys Leu Leu Lys Lys Le - #u Thr Ser His Leu Pro               255              - #   260              - #   265                          - - AAG GCC CGG GTG GTG GCC TGC TTC TGT GAG GG - #C ATG ACG GTG AGA GGT         1224                                                                       Lys Ala Arg Val Val Ala Cys Phe Cys Glu Gl - #y Met Thr Val Arg Gly           270                 2 - #75                 2 - #80                 2 -      #85                                                                              - - CTG CTG ATG GCC ATG AGG CGC CTG GGT CTA GC - #G GGA GAA TTT CTG        CTT     1272                                                                    Leu Leu Met Ala Met Arg Arg Leu Gly Leu Al - #a Gly Glu Phe Leu Leu                          290  - #               295  - #               300              - - CTG GGC AGT GAT GGC TGG GCT GAC AGG TAT GA - #T GTG ACA GAT GGA TAT         1320                                                                       Leu Gly Ser Asp Gly Trp Ala Asp Arg Tyr As - #p Val Thr Asp Gly Tyr                       305      - #           310      - #           315                  - - CAG CGA GAA GCT GTT GGT GGC ATC ACA ATC AA - #G CTC CAA TCT CCC GAT         1368                                                                       Gln Arg Glu Ala Val Gly Gly Ile Thr Ile Ly - #s Leu Gln Ser Pro Asp                   320          - #       325          - #       330                      - - GTC AAG TGG TTT GAT GAT TAT TAT CTG AAG CT - #C CGG CCA GAA ACA AAC         1416                                                                       Val Lys Trp Phe Asp Asp Tyr Tyr Leu Lys Le - #u Arg Pro Glu Thr Asn               335              - #   340              - #   345                          - - CAC CGA AAC CCT TGG TTT CAA GAA TTT TGG CA - #G CAT CGT TTT CAG TGC         1464                                                                       His Arg Asn Pro Trp Phe Gln Glu Phe Trp Gl - #n His Arg Phe Gln Cys           350                 3 - #55                 3 - #60                 3 -      #65                                                                              - - CGA CTG GAA GCG TTT CCA CAG GAG AAC AGC AA - #A TAC AAC AAG ACT        TGC     1512                                                                    Arg Leu Glu Ala Phe Pro Gln Glu Asn Ser Ly - #s Tyr Asn Lys Thr Cys                          370  - #               375  - #               380              - - AAT AGT TCT CTG ACT CTG AAA ACA CAT CAT GT - #T CAG GAT TCC AAA ATG         1560                                                                       Asn Ser Ser Leu Thr Leu Lys Thr His His Va - #l Gln Asp Ser Lys Met                       385      - #           390      - #           395                  - - GGA TTT GTG ATC AAC GCC ATC TAT TCG ATG GC - #C TAT GGG CTC CAC AAC         1608                                                                       Gly Phe Val Ile Asn Ala Ile Tyr Ser Met Al - #a Tyr Gly Leu His Asn                   400          - #       405          - #       410                      - - ATG CAG ATG TCC CTC TGC CCA GGC TAT GCA GG - #A CTC TGT GAT GCC ATG         1656                                                                       Met Gln Met Ser Leu Cys Pro Gly Tyr Ala Gl - #y Leu Cys Asp Ala Met               415              - #   420              - #   425                          - - AAG CCA ATT GAT GGA CGG AAA CTT TTG GAG TC - #C CTG ATG AAA ACC AAT         1704                                                                       Lys Pro Ile Asp Gly Arg Lys Leu Leu Glu Se - #r Leu Met Lys Thr Asn           430                 4 - #35                 4 - #40                 4 -      #45                                                                              - - TTT ACT GGG GTT TCT GGA GAT ACG ATC CTA TT - #C GAT GAG AAT GGA        GAC     1752                                                                    Phe Thr Gly Val Ser Gly Asp Thr Ile Leu Ph - #e Asp Glu Asn Gly Asp                          450  - #               455  - #               460              - - TCT CCA GGA AGG TAT GAA ATA ATG AAT TTC AA - #G GAA ATG GGA AAA GAT         1800                                                                       Ser Pro Gly Arg Tyr Glu Ile Met Asn Phe Ly - #s Glu Met Gly Lys Asp                       465      - #           470      - #           475                  - - TAC TTT GAT TAT ATC AAC GTT GGA AGT TGG GA - #C AAT GGA GAA TTA AAA         1848                                                                       Tyr Phe Asp Tyr Ile Asn Val Gly Ser Trp As - #p Asn Gly Glu Leu Lys                   480          - #       485          - #       490                      - - ATG GAT GAT GAT GAA GTA TGG TCC AAG AAA AG - #C AAC ATC ATC AGA TCT         1896                                                                       Met Asp Asp Asp Glu Val Trp Ser Lys Lys Se - #r Asn Ile Ile Arg Ser               495              - #   500              - #   505                          - - GTG TGC AGT GAA CCA TGT GAG AAA GGC CAG AT - #C AAG GTG ATC CGA AAG         1944                                                                       Val Cys Ser Glu Pro Cys Glu Lys Gly Gln Il - #e Lys Val Ile Arg Lys           510                 5 - #15                 5 - #20                 5 -      #25                                                                              - - GGA GAA GTC AGC TGT TGT TGG ACC TGT ACA CC - #T TGT AAG GAG AAT        GAG     1992                                                                    Gly Glu Val Ser Cys Cys Trp Thr Cys Thr Pr - #o Cys Lys Glu Asn Glu                          530  - #               535  - #               540              - - TAT GTC TTT GAT GAG TAC ACA TGC AAG GCA TG - #C CAA CTG GGG TCT TGG         2040                                                                       Tyr Val Phe Asp Glu Tyr Thr Cys Lys Ala Cy - #s Gln Leu Gly Ser Trp                       545      - #           550      - #           555                  - - CCC ACT GAT GAT CTC ACA GGT TGT GAC TTG AT - #C CCA GTA CAG TAT CTT         2088                                                                       Pro Thr Asp Asp Leu Thr Gly Cys Asp Leu Il - #e Pro Val Gln Tyr Leu                   560          - #       565          - #       570                      - - CGA TGG GGT GAC CCT GAA CCC ATT GCA GCT GT - #G GTG TTT GCC TGC CTT         2136                                                                       Arg Trp Gly Asp Pro Glu Pro Ile Ala Ala Va - #l Val Phe Ala Cys Leu               575              - #   580              - #   585                          - - GGC CTC CTG GCC ACC CTG TTT GTT ACT GTA GT - #C TTC ATC ATT TAC CGT         2184                                                                       Gly Leu Leu Ala Thr Leu Phe Val Thr Val Va - #l Phe Ile Ile Tyr Arg           590                 5 - #95                 6 - #00                 6 -      #05                                                                              - - GAT ACA CCA GTA GTC AAG TCC TCA AGC AGG GA - #A CTC TGC TAC ATT        ATC     2232                                                                    Asp Thr Pro Val Val Lys Ser Ser Ser Arg Gl - #u Leu Cys Tyr Ile Ile                          610  - #               615  - #               620              - - CTT GCT GGC ATC TGC CTG GGC TAC TTA TGT AC - #C TTC TGC CTC ATT GCG         2280                                                                       Leu Ala Gly Ile Cys Leu Gly Tyr Leu Cys Th - #r Phe Cys Leu Ile Ala                       625      - #           630      - #           635                  - - AAG CCC AAA CAG ATT TAC TGC TAC CTT CAG AG - #A ATT GGC ATT GGT CTC         2328                                                                       Lys Pro Lys Gln Ile Tyr Cys Tyr Leu Gln Ar - #g Ile Gly Ile Gly Leu                   640          - #       645          - #       650                      - - TCC CCA GCC ATG AGC TAC TCA GCC CTT GTA AC - #A AAG ACC AAC CGT ATT         2376                                                                       Ser Pro Ala Met Ser Tyr Ser Ala Leu Val Th - #r Lys Thr Asn Arg Ile               655              - #   660              - #   665                          - - GCA AGG ATC CTG GCT GGC AGC AAG AAG AAG AT - #C TGT ACC CCC AAG CCC         2424                                                                       Ala Arg Ile Leu Ala Gly Ser Lys Lys Lys Il - #e Cys Thr Pro Lys Pro           670                 6 - #75                 6 - #80                 6 -      #85                                                                              - - AGA TTC ATG AGT GCC TGT GCC CAG CTA GTG AT - #T GCT TTC ATT CTC        ATA     2472                                                                    Arg Phe Met Ser Ala Cys Ala Gln Leu Val Il - #e Ala Phe Ile Leu Ile                          690  - #               695  - #               700              - - TGC ATC CAG TTG GGC ATC ATC GTT GCC CTC TT - #T ATA ATG GAG CCT CCT         2520                                                                       Cys Ile Gln Leu Gly Ile Ile Val Ala Leu Ph - #e Ile Met Glu Pro Pro                       705      - #           710      - #           715                  - - GAC ATA ATG CAT GAC TAC CCA AGC ATT CGA GA - #A GTC TAC CTG ATC TGT         2568                                                                       Asp Ile Met His Asp Tyr Pro Ser Ile Arg Gl - #u Val Tyr Leu Ile Cys                   720          - #       725          - #       730                      - - AAC ACC ACC AAC CTA GGA GTT GTC ACT CCA CT - #T GGA AAC AAT GGA TTG         2616                                                                       Asn Thr Thr Asn Leu Gly Val Val Thr Pro Le - #u Gly Asn Asn Gly Leu               735              - #   740              - #   745                          - - TTG ATT TTG AGC TGC ACC TTC TAT GCG TTC AA - #G ACC AGA AAT GTT CCA         2664                                                                       Leu Ile Leu Ser Cys Thr Phe Tyr Ala Phe Ly - #s Thr Arg Asn Val Pro           750                 7 - #55                 7 - #60                 7 -      #65                                                                              - - GCT AAC TTC CCC GAG GCC AAG TAT ATC GCC TT - #C ACA ATG TAC ACG        ACC     2712                                                                    Ala Asn Phe Pro Glu Ala Lys Tyr Ile Ala Ph - #e Thr Met Tyr Thr Thr                          770  - #               775  - #               780              - - TGC ATT ATA TGG CTA GCT TTT GTT CCA ATC TA - #C TTT GGC AGC AAC TAC         2760                                                                       Cys Ile Ile Trp Leu Ala Phe Val Pro Ile Ty - #r Phe Gly Ser Asn Tyr                       785      - #           790      - #           795                  - - AAA ATC ATC ACC ATG TGT TTC TCG GTC AGC CT - #C AGT GCC ACA GTG GCC         2808                                                                       Lys Ile Ile Thr Met Cys Phe Ser Val Ser Le - #u Ser Ala Thr Val Ala                   800          - #       805          - #       810                      - - CTA GGC TGC ATG TTT GTG CCG AAG GTG TAC AT - #C ATC CTG GCC AAA CCA         2856                                                                       Leu Gly Cys Met Phe Val Pro Lys Val Tyr Il - #e Ile Leu Ala Lys Pro               815              - #   820              - #   825                          - - GAG AGA AAC GTG CGC AGC GCC TTC ACC ACA TC - #T ACC GTG GTG CGC ATG         2904                                                                       Glu Arg Asn Val Arg Ser Ala Phe Thr Thr Se - #r Thr Val Val Arg Met           830                 8 - #35                 8 - #40                 8 -      #45                                                                              - - CAT GTA GGG GAT GGC AAG TCA TCC TCC GCA GC - #C AGC AGA TCC AGC        AGC     2952                                                                    His Val Gly Asp Gly Lys Ser Ser Ser Ala Al - #a Ser Arg Ser Ser Ser                          850  - #               855  - #               860              - - CTA GTC AAC CTG TGG AAG AGA AGG GGC TCC TC - #T GGG GAA ACC TTA AGG         3000                                                                       Leu Val Asn Leu Trp Lys Arg Arg Gly Ser Se - #r Gly Glu Thr Leu Arg                       865      - #           870      - #           875                  - - TAC AAA GAC AGG AGA CTG GCC CAG CAC AAG TC - #G GAA ATA GAG TGT TTC         3048                                                                       Tyr Lys Asp Arg Arg Leu Ala Gln His Lys Se - #r Glu Ile Glu Cys Phe                   880          - #       885          - #       890                      - - ACC CCC AAA GGG AGT ATG GGG AAT GGT GGG AG - #A GCA ACA ATG AGC AGT         3096                                                                       Thr Pro Lys Gly Ser Met Gly Asn Gly Gly Ar - #g Ala Thr Met Ser Ser               895              - #   900              - #   905                          - - TCC AAT GGA AAA TCC GTC ACG TGG GCC CAG AA - #T GAG AAG AGC AGC CGG         3144                                                                       Ser Asn Gly Lys Ser Val Thr Trp Ala Gln As - #n Glu Lys Ser Ser Arg           910                 9 - #15                 9 - #20                 9 -      #25                                                                              - - GGG CAG CAC CTG TGG CAG CGC CTG TCC ATC CA - #C ATC AAC AAG AAA        GAA     3192                                                                    Gly Gln His Leu Trp Gln Arg Leu Ser Ile Hi - #s Ile Asn Lys Lys Glu                          930  - #               935  - #               940              - - AAC CCC AAC CAA ACG GCC GTC ATC AAG CCC TT - #C CCC AAG AGC ACG GAG         3240                                                                       Asn Pro Asn Gln Thr Ala Val Ile Lys Pro Ph - #e Pro Lys Ser Thr Glu                       945      - #           950      - #           955                  - - AGC CGT GGC CTG GGC GCT GGC GCT GGC GCA GG - #C GGG AGC GCT GGG GGC         3288                                                                       Ser Arg Gly Leu Gly Ala Gly Ala Gly Ala Gl - #y Gly Ser Ala Gly Gly                   960          - #       965          - #       970                      - - GTG GGG GCC ACG GGC GGT GCG GGC TGC GCA GG - #C GCC GGC CCA GGC GGG         3336                                                                       Val Gly Ala Thr Gly Gly Ala Gly Cys Ala Gl - #y Ala Gly Pro Gly Gly               975              - #   980              - #   985                          - - CCC GAG TCC CCA GAC GCC GGC CCC AAG GCG CT - #G TAT GAT GTG GCC GAG         3384                                                                       Pro Glu Ser Pro Asp Ala Gly Pro Lys Ala Le - #u Tyr Asp Val Ala Glu           990                 9 - #95                 1 - #000                1005       - - GCT GAG GAG CAC TTC CCG GCG CCC GCG CGG CC - #G CGC TCA CCG TCG CCC         3432                                                                       Ala Glu Glu His Phe Pro Ala Pro Ala Arg Pr - #o Arg Ser Pro Ser Pro                           1010 - #               1015  - #              1020             - - ATC AGC ACG CTG AGC CAC CGC GCG GGC TCG GC - #C AGC CGC ACG GAC GAC         3480                                                                       Ile Ser Thr Leu Ser His Arg Ala Gly Ser Al - #a Ser Arg Thr Asp Asp                       1025     - #           1030      - #          1035                 - - GAT GTG CCG TCG CTG CAC TCG GAG CCT GTG GC - #G CGC AGC AGC TCC TCG         3528                                                                       Asp Val Pro Ser Leu His Ser Glu Pro Val Al - #a Arg Ser Ser Ser Ser                   1040         - #       1045          - #      1050                     - - CAG GGC TCC CTC ATG GAG CAG ATC AGC AGT GT - #G GTC ACC CGC TTC ACG         3576                                                                       Gln Gly Ser Leu Met Glu Gln Ile Ser Ser Va - #l Val Thr Arg Phe Thr               1055             - #   1060              - #  1065                         - - GCC AAC ATC AGC GAG CTC AAC TCC ATG ATG CT - #G TCC ACC GCG GCC CCC         3624                                                                       Ala Asn Ile Ser Glu Leu Asn Ser Met Met Le - #u Ser Thr Ala Ala Pro           1070                1075 - #                1080 - #               1085        - - AGC CCC GGC GTC GGC GCC CCG CTC TGC TCG TC - #C TAC CTG ATC CCC AAA         3672                                                                       Ser Pro Gly Val Gly Ala Pro Leu Cys Ser Se - #r Tyr Leu Ile Pro Lys                           1090 - #               1095  - #              1100             - - GAG ATC CAG TTG CCC ACG ACC ATG ACG ACC TT - #T GCC GAA ATC CAG CCT         3720                                                                       Glu Ile Gln Leu Pro Thr Thr Met Thr Thr Ph - #e Ala Glu Ile Gln Pro                       1105     - #           1110      - #          1115                 - - CTG CCG GCC ATC GAA GTC ACG GGC GGC GCT CA - #G CCC GCG GCA GGG GCG         3768                                                                       Leu Pro Ala Ile Glu Val Thr Gly Gly Ala Gl - #n Pro Ala Ala Gly Ala                   1120         - #       1125          - #      1130                     - - CAG GCG GCT GGG GAC GCG GCC CGG GAG AGC CC - #C GCG GCC GGT CCC GAG         3816                                                                       Gln Ala Ala Gly Asp Ala Ala Arg Glu Ser Pr - #o Ala Ala Gly Pro Glu               1135             - #   1140              - #  1145                         - - GCT GCG GCC GCC AAG CCA GAC CTG GAG GAG CT - #G GTG GCT CTC ACC CCG         3864                                                                       Ala Ala Ala Ala Lys Pro Asp Leu Glu Glu Le - #u Val Ala Leu Thr Pro           1150                1155 - #                1160 - #               1165        - - CCG TCC CCC TTC AGA GAC TCG GTG GAC TCG GG - #G AGC ACA ACC CCC AAC         3912                                                                       Pro Ser Pro Phe Arg Asp Ser Val Asp Ser Gl - #y Ser Thr Thr Pro Asn                           1170 - #               1175  - #              1180             - - TCG CCA GTG TCC GAG TCG GCC CTC TGT ATC CC - #G TCG TCT CCC AAA TAT         3960                                                                       Ser Pro Val Ser Glu Ser Ala Leu Cys Ile Pr - #o Ser Ser Pro Lys Tyr                       1185     - #           1190      - #          1195                 - - GAC ACT CTT ATC ATA AGA GAT TAC ACT CAG AG - #C TCC TCG TCG TTG         TGAATGTC4015                                                                    Asp Thr Leu Ile Ile Arg Asp Tyr Thr Gln Se - #r Ser Ser Ser Leu                      1200         - #       1205          - #      1210                     - - TGGAAAGCAC GCCGGCCTGC GCGTGCGGAG CGGAGCCCCC CGTGTTCACA CA -             #CACACAAT   4075                                                                 - - GGCAAGCATA GTCGCCTGGT TACGGCCCAG GGGGAAGATG CCAAGGGCAC CC -            #CTTAATGG   4135                                                                 - - AAACACGAGA TCAGTAGTGC TATCTCATGA CAACCGACGA AGAAAC   - #                   4181                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1212 amino - #acids                                               (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Met Val Leu Leu Leu Ile Leu Ser Val Leu Le - #u Trp Lys Glu Asp Val        1               5 - #                 10 - #                 15              - - Arg Gly Ser Ala Gln Ser Ser Glu Arg Arg Va - #l Val Ala His Met Pro                   20     - #             25     - #             30                  - - Gly Asp Ile Ile Ile Gly Ala Leu Phe Ser Va - #l His His Gln Pro Thr               35         - #         40         - #         45                      - - Val Asp Lys Val His Glu Arg Lys Cys Gly Al - #a Val Arg Glu Gln Tyr           50             - #     55             - #     60                          - - Gly Ile Gln Arg Val Glu Ala Met Leu His Th - #r Leu Glu Arg Ile Asn       65                 - # 70                 - # 75                 - # 80       - - Ser Asp Pro Thr Leu Leu Pro Asn Ile Thr Le - #u Gly Cys Glu Ile Arg                       85 - #                 90 - #                 95              - - Asp Ser Cys Trp His Ser Ala Val Ala Leu Gl - #u Gln Ser Ile Glu Phe                  100      - #           105      - #           110                  - - Ile Arg Asp Ser Leu Ile Ser Ser Glu Glu Gl - #u Glu Gly Leu Val Arg              115          - #       120          - #       125                      - - Cys Val Asp Gly Ser Ser Ser Ser Phe Arg Se - #r Lys Lys Pro Ile Val          130              - #   135              - #   140                          - - Gly Val Ile Gly Pro Gly Ser Ser Ser Val Al - #a Ile Gln Val Gln Asn      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Leu Gln Leu Phe Asn Ile Pro Gln Ile Al - #a Tyr Ser Ala Thr        Ser                                                                                             165  - #               170  - #               175             - - Met Asp Leu Ser Asp Lys Thr Leu Phe Lys Ty - #r Phe Met Arg Val Val                  180      - #           185      - #           190                  - - Pro Ser Asp Ala Gln Gln Ala Arg Ala Met Va - #l Asp Ile Val Lys Arg              195          - #       200          - #       205                      - - Tyr Asn Trp Thr Tyr Val Ser Ala Val His Th - #r Glu Gly Asn Tyr Gly          210              - #   215              - #   220                          - - Glu Ser Gly Met Glu Ala Ser Lys Asp Met Se - #r Ala Lys Glu Gly Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Cys Ile Ala His Ser Tyr Lys Ile Tyr Ser As - #n Ala Gly Glu Gln        Ser                                                                                             245  - #               250  - #               255             - - Phe Asp Lys Leu Leu Lys Lys Leu Thr Ser Hi - #s Leu Pro Lys Ala Arg                  260      - #           265      - #           270                  - - Val Val Ala Cys Phe Cys Glu Gly Met Thr Va - #l Arg Gly Leu Leu Met              275          - #       280          - #       285                      - - Ala Met Arg Arg Leu Gly Leu Ala Gly Glu Ph - #e Leu Leu Leu Gly Ser          290              - #   295              - #   300                          - - Asp Gly Trp Ala Asp Arg Tyr Asp Val Thr As - #p Gly Tyr Gln Arg Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ala Val Gly Gly Ile Thr Ile Lys Leu Gln Se - #r Pro Asp Val Lys        Trp                                                                                             325  - #               330  - #               335             - - Phe Asp Asp Tyr Tyr Leu Lys Leu Arg Pro Gl - #u Thr Asn His Arg Asn                  340      - #           345      - #           350                  - - Pro Trp Phe Gln Glu Phe Trp Gln His Arg Ph - #e Gln Cys Arg Leu Glu              355          - #       360          - #       365                      - - Ala Phe Pro Gln Glu Asn Ser Lys Tyr Asn Ly - #s Thr Cys Asn Ser Ser          370              - #   375              - #   380                          - - Leu Thr Leu Lys Thr His His Val Gln Asp Se - #r Lys Met Gly Phe Val      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ile Asn Ala Ile Tyr Ser Met Ala Tyr Gly Le - #u His Asn Met Gln        Met                                                                                             405  - #               410  - #               415             - - Ser Leu Cys Pro Gly Tyr Ala Gly Leu Cys As - #p Ala Met Lys Pro Ile                  420      - #           425      - #           430                  - - Asp Gly Arg Lys Leu Leu Glu Ser Leu Met Ly - #s Thr Asn Phe Thr Gly              435          - #       440          - #       445                      - - Val Ser Gly Asp Thr Ile Leu Phe Asp Glu As - #n Gly Asp Ser Pro Gly          450              - #   455              - #   460                          - - Arg Tyr Glu Ile Met Asn Phe Lys Glu Met Gl - #y Lys Asp Tyr Phe Asp      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Tyr Ile Asn Val Gly Ser Trp Asp Asn Gly Gl - #u Leu Lys Met Asp        Asp                                                                                             485  - #               490  - #               495             - - Asp Glu Val Trp Ser Lys Lys Ser Asn Ile Il - #e Arg Ser Val Cys Ser                  500      - #           505      - #           510                  - - Glu Pro Cys Glu Lys Gly Gln Ile Lys Val Il - #e Arg Lys Gly Glu Val              515          - #       520          - #       525                      - - Ser Cys Cys Trp Thr Cys Thr Pro Cys Lys Gl - #u Asn Glu Tyr Val Phe          530              - #   535              - #   540                          - - Asp Glu Tyr Thr Cys Lys Ala Cys Gln Leu Gl - #y Ser Trp Pro Thr Asp      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Asp Leu Thr Gly Cys Asp Leu Ile Pro Val Gl - #n Tyr Leu Arg Trp        Gly                                                                                             565  - #               570  - #               575             - - Asp Pro Glu Pro Ile Ala Ala Val Val Phe Al - #a Cys Leu Gly Leu Leu                  580      - #           585      - #           590                  - - Ala Thr Leu Phe Val Thr Val Val Phe Ile Il - #e Tyr Arg Asp Thr Pro              595          - #       600          - #       605                      - - Val Val Lys Ser Ser Ser Arg Glu Leu Cys Ty - #r Ile Ile Leu Ala Gly          610              - #   615              - #   620                          - - Ile Cys Leu Gly Tyr Leu Cys Thr Phe Cys Le - #u Ile Ala Lys Pro Lys      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Gln Ile Tyr Cys Tyr Leu Gln Arg Ile Gly Il - #e Gly Leu Ser Pro        Ala                                                                                             645  - #               650  - #               655             - - Met Ser Tyr Ser Ala Leu Val Thr Lys Thr As - #n Arg Ile Ala Arg Ile                  660      - #           665      - #           670                  - - Leu Ala Gly Ser Lys Lys Lys Ile Cys Thr Pr - #o Lys Pro Arg Phe Met              675          - #       680          - #       685                      - - Ser Ala Cys Ala Gln Leu Val Ile Ala Phe Il - #e Leu Ile Cys Ile Gln          690              - #   695              - #   700                          - - Leu Gly Ile Ile Val Ala Leu Phe Ile Met Gl - #u Pro Pro Asp Ile Met      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - His Asp Tyr Pro Ser Ile Arg Glu Val Tyr Le - #u Ile Cys Asn Thr        Thr                                                                                             725  - #               730  - #               735             - - Asn Leu Gly Val Val Thr Pro Leu Gly Asn As - #n Gly Leu Leu Ile Leu                  740      - #           745      - #           750                  - - Ser Cys Thr Phe Tyr Ala Phe Lys Thr Arg As - #n Val Pro Ala Asn Phe              755          - #       760          - #       765                      - - Pro Glu Ala Lys Tyr Ile Ala Phe Thr Met Ty - #r Thr Thr Cys Ile Ile          770              - #   775              - #   780                          - - Trp Leu Ala Phe Val Pro Ile Tyr Phe Gly Se - #r Asn Tyr Lys Ile Ile      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Thr Met Cys Phe Ser Val Ser Leu Ser Ala Th - #r Val Ala Leu Gly        Cys                                                                                             805  - #               810  - #               815             - - Met Phe Val Pro Lys Val Tyr Ile Ile Leu Al - #a Lys Pro Glu Arg Asn                  820      - #           825      - #           830                  - - Val Arg Ser Ala Phe Thr Thr Ser Thr Val Va - #l Arg Met His Val Gly              835          - #       840          - #       845                      - - Asp Gly Lys Ser Ser Ser Ala Ala Ser Arg Se - #r Ser Ser Leu Val Asn          850              - #   855              - #   860                          - - Leu Trp Lys Arg Arg Gly Ser Ser Gly Glu Th - #r Leu Arg Tyr Lys Asp      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - Arg Arg Leu Ala Gln His Lys Ser Glu Ile Gl - #u Cys Phe Thr Pro        Lys                                                                                             885  - #               890  - #               895             - - Gly Ser Met Gly Asn Gly Gly Arg Ala Thr Me - #t Ser Ser Ser Asn Gly                  900      - #           905      - #           910                  - - Lys Ser Val Thr Trp Ala Gln Asn Glu Lys Se - #r Ser Arg Gly Gln His              915          - #       920          - #       925                      - - Leu Trp Gln Arg Leu Ser Ile His Ile Asn Ly - #s Lys Glu Asn Pro Asn          930              - #   935              - #   940                          - - Gln Thr Ala Val Ile Lys Pro Phe Pro Lys Se - #r Thr Glu Ser Arg Gly      945                 9 - #50                 9 - #55                 9 -      #60                                                                              - - Leu Gly Ala Gly Ala Gly Ala Gly Gly Ser Al - #a Gly Gly Val Gly        Ala                                                                                             965  - #               970  - #               975             - - Thr Gly Gly Ala Gly Cys Ala Gly Ala Gly Pr - #o Gly Gly Pro Glu Ser                  980      - #           985      - #           990                  - - Pro Asp Ala Gly Pro Lys Ala Leu Tyr Asp Va - #l Ala Glu Ala Glu Glu              995          - #       1000          - #      1005                     - - His Phe Pro Ala Pro Ala Arg Pro Arg Ser Pr - #o Ser Pro Ile Ser Thr          1010             - #   1015              - #  1020                         - - Leu Ser His Arg Ala Gly Ser Ala Ser Arg Th - #r Asp Asp Asp Val Pro      1025                1030 - #                1035 - #               1040        - - Ser Leu His Ser Glu Pro Val Ala Arg Ser Se - #r Ser Ser Gln Gly Ser                      1045 - #               1050  - #              1055             - - Leu Met Glu Gln Ile Ser Ser Val Val Thr Ar - #g Phe Thr Ala Asn Ile                  1060     - #           1065      - #          1070                 - - Ser Glu Leu Asn Ser Met Met Leu Ser Thr Al - #a Ala Pro Ser Pro Gly              1075         - #       1080          - #      1085                     - - Val Gly Ala Pro Leu Cys Ser Ser Tyr Leu Il - #e Pro Lys Glu Ile Gln          1090             - #   1095              - #  1100                         - - Leu Pro Thr Thr Met Thr Thr Phe Ala Glu Il - #e Gln Pro Leu Pro Ala      1105                1110 - #                1115 - #               1120        - - Ile Glu Val Thr Gly Gly Ala Gln Pro Ala Al - #a Gly Ala Gln Ala Ala                      1125 - #               1130  - #              1135             - - Gly Asp Ala Ala Arg Glu Ser Pro Ala Ala Gl - #y Pro Glu Ala Ala Ala                  1140     - #           1145      - #          1150                 - - Ala Lys Pro Asp Leu Glu Glu Leu Val Ala Le - #u Thr Pro Pro Ser Pro              1155         - #       1160          - #      1165                     - - Phe Arg Asp Ser Val Asp Ser Gly Ser Thr Th - #r Pro Asn Ser Pro Val          1170             - #   1175              - #  1180                         - - Ser Glu Ser Ala Leu Cys Ile Pro Ser Ser Pr - #o Lys Tyr Asp Thr Leu      1185                1190 - #                1195 - #               1200        - - Ile Ile Arg Asp Tyr Thr Gln Ser Ser Ser Se - #r Leu                                      1205 - #               1210                                    - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3282 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                   - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 370..3003                                                       (D) OTHER INFORMATION: - #/product= "HUMAN MGLUR5C"                                /note= - #"Variant of MGLUR5A with truncated 3' end."           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - CAGCTCGGCT GTTCTGCGCA CGCTGAGCGG AGGGAATGAG CTTGAGATCA TC -             #TTGGGGGG     60                                                                 - - GAAGCCGGGG ACTGGAGAGG CCGGCTCTGC CCTGCTGATC CCCGTGGCCC AA -            #CTTTTCGG    120                                                                 - - GGGGCTAGCT AGACCGAGTC TCACTGCTCG CAGCGCAGCC AACAGGGGGG TT -            #TAGAAGAT    180                                                                 - - CATGACCACA TGGATCATCT AACTAAATGG TACATGGGGA CAAAATGGTC CT -            #TTAGAAAA    240                                                                 - - TACATCTGAA TTGCTGGCTA ATTTCTTGAT TTGCGACTCA ACGTAGGACA TC -            #GCTTGTTC    300                                                                 - - GTAGCTATCA GAACCCTCCT GAATTTTCCC CACCATGCTA TCTTTATTGG CT -            #TGAACTCC    360                                                                 - - TTTCCTAAA ATG GTC CTT CTG TTG ATC CTG TCA GTC - # TTA CTT TGG AAA            408                                                                                Met Val Leu Leu Leu - #Ile Leu Ser Val Leu Leu Trp Lys                          1      - #         5         - #         10                        - - GAA GAT GTC CGT GGG AGT GCA CAG TCC AGT GA - #G AGG AGG GTG GTG GCT          456                                                                       Glu Asp Val Arg Gly Ser Ala Gln Ser Ser Gl - #u Arg Arg Val Val Ala                15             - #     20             - #     25                          - - CAC ATG CCG GGT GAC ATC ATT ATT GGA GCT CT - #C TTT TCT GTT CAT CAC          504                                                                       His Met Pro Gly Asp Ile Ile Ile Gly Ala Le - #u Phe Ser Val His His            30                 - # 35                 - # 40                 - # 45       - - CAG CCT ACT GTG GAC AAA GTT CAT GAG AGG AA - #G TGT GGG GCG GTC CGT          552                                                                       Gln Pro Thr Val Asp Lys Val His Glu Arg Ly - #s Cys Gly Ala Val Arg                            50 - #                 55 - #                 60              - - GAA CAG TAT GGC ATT CAG AGA GTG GAG GCC AT - #G CTG CAT ACC CTG GAA          600                                                                       Glu Gln Tyr Gly Ile Gln Arg Val Glu Ala Me - #t Leu His Thr Leu Glu                        65     - #             70     - #             75                  - - AGG ATC AAT TCA GAC CCC ACA CTC TTG CCC AA - #C ATC ACA CTG GGC TGT          648                                                                       Arg Ile Asn Ser Asp Pro Thr Leu Leu Pro As - #n Ile Thr Leu Gly Cys                    80         - #         85         - #         90                      - - GAG ATA AGG GAC TCC TGC TGG CAT TCG GCT GT - #G GCC CTA GAG CAG AGC          696                                                                       Glu Ile Arg Asp Ser Cys Trp His Ser Ala Va - #l Ala Leu Glu Gln Ser                95             - #    100             - #    105                          - - ATT GAG TTC ATA AGA GAT TCC CTC ATT TCT TC - #A GAA GAG GAA GAA GGC          744                                                                       Ile Glu Phe Ile Arg Asp Ser Leu Ile Ser Se - #r Glu Glu Glu Glu Gly           110                 1 - #15                 1 - #20                 1 -      #25                                                                              - - TTG GTA CGC TGT GTG GAT GGC TCC TCC TCT TC - #C TTC CGC TCC AAG        AAG      792                                                                    Leu Val Arg Cys Val Asp Gly Ser Ser Ser Se - #r Phe Arg Ser Lys Lys                          130  - #               135  - #               140              - - CCC ATA GTA GGG GTC ATT GGG CCT GGC TCC AG - #T TCT GTA GCC ATT CAG          840                                                                       Pro Ile Val Gly Val Ile Gly Pro Gly Ser Se - #r Ser Val Ala Ile Gln                       145      - #           150      - #           155                  - - GTC CAG AAT TTG CTC CAG CTT TTC AAC ATA CC - #T CAG ATT GCT TAC TCA          888                                                                       Val Gln Asn Leu Leu Gln Leu Phe Asn Ile Pr - #o Gln Ile Ala Tyr Ser                   160          - #       165          - #       170                      - - GCA ACC AGC ATG GAT CTG AGT GAC AAG ACT CT - #G TTC AAA TAT TTC ATG          936                                                                       Ala Thr Ser Met Asp Leu Ser Asp Lys Thr Le - #u Phe Lys Tyr Phe Met               175              - #   180              - #   185                          - - AGG GTT GTG CCT TCA GAT GCT CAG CAG GCA AG - #G GCC ATG GTG GAC ATA          984                                                                       Arg Val Val Pro Ser Asp Ala Gln Gln Ala Ar - #g Ala Met Val Asp Ile           190                 1 - #95                 2 - #00                 2 -      #05                                                                              - - GTG AAG AGG TAC AAC TGG ACC TAT GTA TCA GC - #C GTG CAC ACA GAA        GGC     1032                                                                    Val Lys Arg Tyr Asn Trp Thr Tyr Val Ser Al - #a Val His Thr Glu Gly                          210  - #               215  - #               220              - - AAC TAT GGA GAA AGT GGG ATG GAA GCC TCC AA - #A GAT ATG TCA GCG AAG         1080                                                                       Asn Tyr Gly Glu Ser Gly Met Glu Ala Ser Ly - #s Asp Met Ser Ala Lys                       225      - #           230      - #           235                  - - GAA GGG ATT TGC ATC GCC CAC TCT TAC AAA AT - #C TAC AGT AAT GCA GGG         1128                                                                       Glu Gly Ile Cys Ile Ala His Ser Tyr Lys Il - #e Tyr Ser Asn Ala Gly                   240          - #       245          - #       250                      - - GAG CAG AGC TTT GAT AAG CTG CTG AAG AAG CT - #C ACA AGT CAC TTG CCC         1176                                                                       Glu Gln Ser Phe Asp Lys Leu Leu Lys Lys Le - #u Thr Ser His Leu Pro               255              - #   260              - #   265                          - - AAG GCC CGG GTG GTG GCC TGC TTC TGT GAG GG - #C ATG ACG GTG AGA GGT         1224                                                                       Lys Ala Arg Val Val Ala Cys Phe Cys Glu Gl - #y Met Thr Val Arg Gly           270                 2 - #75                 2 - #80                 2 -      #85                                                                              - - CTG CTG ATG GCC ATG AGG CGC CTG GGT CTA GC - #G GGA GAA TTT CTG        CTT     1272                                                                    Leu Leu Met Ala Met Arg Arg Leu Gly Leu Al - #a Gly Glu Phe Leu Leu                          290  - #               295  - #               300              - - CTG GGC AGT GAT GGC TGG GCT GAC AGG TAT GA - #T GTG ACA GAT GGA TAT         1320                                                                       Leu Gly Ser Asp Gly Trp Ala Asp Arg Tyr As - #p Val Thr Asp Gly Tyr                       305      - #           310      - #           315                  - - CAG CGA GAA GCT GTT GGT GGC ATC ACA ATC AA - #G CTC CAA TCT CCC GAT         1368                                                                       Gln Arg Glu Ala Val Gly Gly Ile Thr Ile Ly - #s Leu Gln Ser Pro Asp                   320          - #       325          - #       330                      - - GTC AAG TGG TTT GAT GAT TAT TAT CTG AAG CT - #C CGG CCA GAA ACA AAC         1416                                                                       Val Lys Trp Phe Asp Asp Tyr Tyr Leu Lys Le - #u Arg Pro Glu Thr Asn               335              - #   340              - #   345                          - - CAC CGA AAC CCT TGG TTT CAA GAA TTT TGG CA - #G CAT CGT TTT CAG TGC         1464                                                                       His Arg Asn Pro Trp Phe Gln Glu Phe Trp Gl - #n His Arg Phe Gln Cys           350                 3 - #55                 3 - #60                 3 -      #65                                                                              - - CGA CTG GAA GCG TTT CCA CAG GAG AAC AGC AA - #A TAC AAC AAG ACT        TGC     1512                                                                    Arg Leu Glu Ala Phe Pro Gln Glu Asn Ser Ly - #s Tyr Asn Lys Thr Cys                          370  - #               375  - #               380              - - AAT AGT TCT CTG ACT CTG AAA ACA CAT CAT GT - #T CAG GAT TCC AAA ATG         1560                                                                       Asn Ser Ser Leu Thr Leu Lys Thr His His Va - #l Gln Asp Ser Lys Met                       385      - #           390      - #           395                  - - GGA TTT GTG ATC AAC GCC ATC TAT TCG ATG GC - #C TAT GGG CTC CAC AAC         1608                                                                       Gly Phe Val Ile Asn Ala Ile Tyr Ser Met Al - #a Tyr Gly Leu His Asn                   400          - #       405          - #       410                      - - ATG CAG ATG TCC CTC TGC CCA GGC TAT GCA GG - #A CTC TGT GAT GCC ATG         1656                                                                       Met Gln Met Ser Leu Cys Pro Gly Tyr Ala Gl - #y Leu Cys Asp Ala Met               415              - #   420              - #   425                          - - AAG CCA ATT GAT GGA CGG AAA CTT TTG GAG TC - #C CTG ATG AAA ACC AAT         1704                                                                       Lys Pro Ile Asp Gly Arg Lys Leu Leu Glu Se - #r Leu Met Lys Thr Asn           430                 4 - #35                 4 - #40                 4 -      #45                                                                              - - TTT ACT GGG GTT TCT GGA GAT ACG ATC CTA TT - #C GAT GAG AAT GGA        GAC     1752                                                                    Phe Thr Gly Val Ser Gly Asp Thr Ile Leu Ph - #e Asp Glu Asn Gly Asp                          450  - #               455  - #               460              - - TCT CCA GGA AGG TAT GAA ATA ATG AAT TTC AA - #G GAA ATG GGA AAA GAT         1800                                                                       Ser Pro Gly Arg Tyr Glu Ile Met Asn Phe Ly - #s Glu Met Gly Lys Asp                       465      - #           470      - #           475                  - - TAC TTT GAT TAT ATC AAC GTT GGA AGT TGG GA - #C AAT GGA GAA TTA AAA         1848                                                                       Tyr Phe Asp Tyr Ile Asn Val Gly Ser Trp As - #p Asn Gly Glu Leu Lys                   480          - #       485          - #       490                      - - ATG GAT GAT GAT GAA GTA TGG TCC AAG AAA AG - #C AAC ATC ATC AGA TCT         1896                                                                       Met Asp Asp Asp Glu Val Trp Ser Lys Lys Se - #r Asn Ile Ile Arg Ser               495              - #   500              - #   505                          - - GTG TGC AGT GAA CCA TGT GAG AAA GGC CAG AT - #C AAG GTG ATC CGA AAG         1944                                                                       Val Cys Ser Glu Pro Cys Glu Lys Gly Gln Il - #e Lys Val Ile Arg Lys           510                 5 - #15                 5 - #20                 5 -      #25                                                                              - - GGA GAA GTC AGC TGT TGT TGG ACC TGT ACA CC - #T TGT AAG GAG AAT        GAG     1992                                                                    Gly Glu Val Ser Cys Cys Trp Thr Cys Thr Pr - #o Cys Lys Glu Asn Glu                          530  - #               535  - #               540              - - TAT GTC TTT GAT GAG TAC ACA TGC AAG GCA TG - #C CAA CTG GGG TCT TGG         2040                                                                       Tyr Val Phe Asp Glu Tyr Thr Cys Lys Ala Cy - #s Gln Leu Gly Ser Trp                       545      - #           550      - #           555                  - - CCC ACT GAT GAT CTC ACA GGT TGT GAC TTG AT - #C CCA GTA CAG TAT CTT         2088                                                                       Pro Thr Asp Asp Leu Thr Gly Cys Asp Leu Il - #e Pro Val Gln Tyr Leu                   560          - #       565          - #       570                      - - CGA TGG GGT GAC CCT GAA CCC ATT GCA GCT GT - #G GTG TTT GCC TGC CTT         2136                                                                       Arg Trp Gly Asp Pro Glu Pro Ile Ala Ala Va - #l Val Phe Ala Cys Leu               575              - #   580              - #   585                          - - GGC CTC CTG GCC ACC CTG TTT GTT ACT GTA GT - #C TTC ATC ATT TAC CGT         2184                                                                       Gly Leu Leu Ala Thr Leu Phe Val Thr Val Va - #l Phe Ile Ile Tyr Arg           590                 5 - #95                 6 - #00                 6 -      #05                                                                              - - GAT ACA CCA GTA GTC AAG TCC TCA AGC AGG GA - #A CTC TGC TAC ATT        ATC     2232                                                                    Asp Thr Pro Val Val Lys Ser Ser Ser Arg Gl - #u Leu Cys Tyr Ile Ile                          610  - #               615  - #               620              - - CTT GCT GGC ATC TGC CTG GGC TAC TTA TGT AC - #C TTC TGC CTC ATT GCG         2280                                                                       Leu Ala Gly Ile Cys Leu Gly Tyr Leu Cys Th - #r Phe Cys Leu Ile Ala                       625      - #           630      - #           635                  - - AAG CCC AAA CAG ATT TAC TGC TAC CTT CAG AG - #A ATT GGC ATT GGT CTC         2328                                                                       Lys Pro Lys Gln Ile Tyr Cys Tyr Leu Gln Ar - #g Ile Gly Ile Gly Leu                   640          - #       645          - #       650                      - - TCC CCA GCC ATG AGC TAC TCA GCC CTT GTA AC - #A AAG ACC AAC CGT ATT         2376                                                                       Ser Pro Ala Met Ser Tyr Ser Ala Leu Val Th - #r Lys Thr Asn Arg Ile               655              - #   660              - #   665                          - - GCA AGG ATC CTG GCT GGC AGC AAG AAG AAG AT - #C TGT ACC CCC AAG CCC         2424                                                                       Ala Arg Ile Leu Ala Gly Ser Lys Lys Lys Il - #e Cys Thr Pro Lys Pro           670                 6 - #75                 6 - #80                 6 -      #85                                                                              - - AGA TTC ATG AGT GCC TGT GCC CAG CTA GTG AT - #T GCT TTC ATT CTC        ATA     2472                                                                    Arg Phe Met Ser Ala Cys Ala Gln Leu Val Il - #e Ala Phe Ile Leu Ile                          690  - #               695  - #               700              - - TGC ATC CAG TTG GGC ATC ATC GTT GCC CTC TT - #T ATA ATG GAG CCT CCT         2520                                                                       Cys Ile Gln Leu Gly Ile Ile Val Ala Leu Ph - #e Ile Met Glu Pro Pro                       705      - #           710      - #           715                  - - GAC ATA ATG CAT GAC TAC CCA AGC ATT CGA GA - #A GTC TAC CTG ATC TGT         2568                                                                       Asp Ile Met His Asp Tyr Pro Ser Ile Arg Gl - #u Val Tyr Leu Ile Cys                   720          - #       725          - #       730                      - - AAC ACC ACC AAC CTA GGA GTT GTC ACT CCA CT - #T GGA AAC AAT GGA TTG         2616                                                                       Asn Thr Thr Asn Leu Gly Val Val Thr Pro Le - #u Gly Asn Asn Gly Leu               735              - #   740              - #   745                          - - TTG ATT TTG AGC TGC ACC TTC TAT GCG TTC AA - #G ACC AGA AAT GTT CCA         2664                                                                       Leu Ile Leu Ser Cys Thr Phe Tyr Ala Phe Ly - #s Thr Arg Asn Val Pro           750                 7 - #55                 7 - #60                 7 -      #65                                                                              - - GCT AAC TTC CCC GAG GCC AAG TAT ATC GCC TT - #C ACA ATG TAC ACG        ACC     2712                                                                    Ala Asn Phe Pro Glu Ala Lys Tyr Ile Ala Ph - #e Thr Met Tyr Thr Thr                          770  - #               775  - #               780              - - TGC ATT ATA TGG CTA GCT TTT GTT CCA ATC TA - #C TTT GGC AGC AAC TAC         2760                                                                       Cys Ile Ile Trp Leu Ala Phe Val Pro Ile Ty - #r Phe Gly Ser Asn Tyr                       785      - #           790      - #           795                  - - AAA ATC ATC ACC ATG TGT TTC TCG GTC AGC CT - #C AGT GCC ACA GTG GCC         2808                                                                       Lys Ile Ile Thr Met Cys Phe Ser Val Ser Le - #u Ser Ala Thr Val Ala                   800          - #       805          - #       810                      - - CTA GGC TGC ATG TTT GTG CCG ACG GTG TAC AT - #C ATC CTG GCC AAA CCA         2856                                                                       Leu Gly Cys Met Phe Val Pro Thr Val Tyr Il - #e Ile Leu Ala Lys Pro               815              - #   820              - #   825                          - - GAG AGA AAC GTG CGC AGC GCC TTC ACC ACA TC - #T ACC GTG GTG CGC ATG         2904                                                                       Glu Arg Asn Val Arg Ser Ala Phe Thr Thr Se - #r Thr Val Val Arg Met           830                 8 - #35                 8 - #40                 8 -      #45                                                                              - - CAT GTA GGG GAT GGC AAG TCA TCC TCC GCA GC - #C AGC AGA TCC AGC        AGC     2952                                                                    His Val Gly Asp Gly Lys Ser Ser Ser Ala Al - #a Ser Arg Ser Ser Ser                          850  - #               855  - #               860              - - CTA GTC AAC CTG TGG AAG AGA AGG GGC TCC TC - #T GGG GAA ACC TTA AGG         3000                                                                       Leu Val Asn Leu Trp Lys Arg Arg Gly Ser Se - #r Gly Glu Thr Leu Arg                       865      - #           870      - #           875                  - - TAAAAGTTGT GGGGGCTTAC AGGGATGCTG GCCCCTAAAA CTGGAGCAGA GG -             #CATGTGTT   3060                                                                 - - TCCTGGGTCT TTTAAATGGG AGAAATCTGG GTAAATGACA CCATCTGAGG CA -            #GGGTGACT   3120                                                                 - - TACGGCATGG ACCTCCTCAT AAAATGGTAT TTATGGGGTT AATGGGATGT GG -            #CTCCACTT   3180                                                                 - - ACTTAGCCCA AGTCTAGAAA CATGGAAGTC AAACTCTCTA ATAAAGCAGA GC -            #TACAGCGT   3240                                                                 - - CGGGGGAGTG ACGTTTGACA GGGCAGACAG ACCAGAGTTC AG    - #                      - #3282                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 877 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Met Val Leu Leu Leu Ile Leu Ser Val Leu Le - #u Trp Lys Glu Asp Val        1               5 - #                 10 - #                 15              - - Arg Gly Ser Ala Gln Ser Ser Glu Arg Arg Va - #l Val Ala His Met Pro                   20     - #             25     - #             30                  - - Gly Asp Ile Ile Ile Gly Ala Leu Phe Ser Va - #l His His Gln Pro Thr               35         - #         40         - #         45                      - - Val Asp Lys Val His Glu Arg Lys Cys Gly Al - #a Val Arg Glu Gln Tyr           50             - #     55             - #     60                          - - Gly Ile Gln Arg Val Glu Ala Met Leu His Th - #r Leu Glu Arg Ile Asn       65                 - # 70                 - # 75                 - # 80       - - Ser Asp Pro Thr Leu Leu Pro Asn Ile Thr Le - #u Gly Cys Glu Ile Arg                       85 - #                 90 - #                 95              - - Asp Ser Cys Trp His Ser Ala Val Ala Leu Gl - #u Gln Ser Ile Glu Phe                  100      - #           105      - #           110                  - - Ile Arg Asp Ser Leu Ile Ser Ser Glu Glu Gl - #u Glu Gly Leu Val Arg              115          - #       120          - #       125                      - - Cys Val Asp Gly Ser Ser Ser Ser Phe Arg Se - #r Lys Lys Pro Ile Val          130              - #   135              - #   140                          - - Gly Val Ile Gly Pro Gly Ser Ser Ser Val Al - #a Ile Gln Val Gln Asn      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Leu Gln Leu Phe Asn Ile Pro Gln Ile Al - #a Tyr Ser Ala Thr        Ser                                                                                             165  - #               170  - #               175             - - Met Asp Leu Ser Asp Lys Thr Leu Phe Lys Ty - #r Phe Met Arg Val Val                  180      - #           185      - #           190                  - - Pro Ser Asp Ala Gln Gln Ala Arg Ala Met Va - #l Asp Ile Val Lys Arg              195          - #       200          - #       205                      - - Tyr Asn Trp Thr Tyr Val Ser Ala Val His Th - #r Glu Gly Asn Tyr Gly          210              - #   215              - #   220                          - - Glu Ser Gly Met Glu Ala Ser Lys Asp Met Se - #r Ala Lys Glu Gly Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Cys Ile Ala His Ser Tyr Lys Ile Tyr Ser As - #n Ala Gly Glu Gln        Ser                                                                                             245  - #               250  - #               255             - - Phe Asp Lys Leu Leu Lys Lys Leu Thr Ser Hi - #s Leu Pro Lys Ala Arg                  260      - #           265      - #           270                  - - Val Val Ala Cys Phe Cys Glu Gly Met Thr Va - #l Arg Gly Leu Leu Met              275          - #       280          - #       285                      - - Ala Met Arg Arg Leu Gly Leu Ala Gly Glu Ph - #e Leu Leu Leu Gly Ser          290              - #   295              - #   300                          - - Asp Gly Trp Ala Asp Arg Tyr Asp Val Thr As - #p Gly Tyr Gln Arg Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ala Val Gly Gly Ile Thr Ile Lys Leu Gln Se - #r Pro Asp Val Lys        Trp                                                                                             325  - #               330  - #               335             - - Phe Asp Asp Tyr Tyr Leu Lys Leu Arg Pro Gl - #u Thr Asn His Arg Asn                  340      - #           345      - #           350                  - - Pro Trp Phe Gln Glu Phe Trp Gln His Arg Ph - #e Gln Cys Arg Leu Glu              355          - #       360          - #       365                      - - Ala Phe Pro Gln Glu Asn Ser Lys Tyr Asn Ly - #s Thr Cys Asn Ser Ser          370              - #   375              - #   380                          - - Leu Thr Leu Lys Thr His His Val Gln Asp Se - #r Lys Met Gly Phe Val      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Ile Asn Ala Ile Tyr Ser Met Ala Tyr Gly Le - #u His Asn Met Gln        Met                                                                                             405  - #               410  - #               415             - - Ser Leu Cys Pro Gly Tyr Ala Gly Leu Cys As - #p Ala Met Lys Pro Ile                  420      - #           425      - #           430                  - - Asp Gly Arg Lys Leu Leu Glu Ser Leu Met Ly - #s Thr Asn Phe Thr Gly              435          - #       440          - #       445                      - - Val Ser Gly Asp Thr Ile Leu Phe Asp Glu As - #n Gly Asp Ser Pro Gly          450              - #   455              - #   460                          - - Arg Tyr Glu Ile Met Asn Phe Lys Glu Met Gl - #y Lys Asp Tyr Phe Asp      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Tyr Ile Asn Val Gly Ser Trp Asp Asn Gly Gl - #u Leu Lys Met Asp        Asp                                                                                             485  - #               490  - #               495             - - Asp Glu Val Trp Ser Lys Lys Ser Asn Ile Il - #e Arg Ser Val Cys Ser                  500      - #           505      - #           510                  - - Glu Pro Cys Glu Lys Gly Gln Ile Lys Val Il - #e Arg Lys Gly Glu Val              515          - #       520          - #       525                      - - Ser Cys Cys Trp Thr Cys Thr Pro Cys Lys Gl - #u Asn Glu Tyr Val Phe          530              - #   535              - #   540                          - - Asp Glu Tyr Thr Cys Lys Ala Cys Gln Leu Gl - #y Ser Trp Pro Thr Asp      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Asp Leu Thr Gly Cys Asp Leu Ile Pro Val Gl - #n Tyr Leu Arg Trp        Gly                                                                                             565  - #               570  - #               575             - - Asp Pro Glu Pro Ile Ala Ala Val Val Phe Al - #a Cys Leu Gly Leu Leu                  580      - #           585      - #           590                  - - Ala Thr Leu Phe Val Thr Val Val Phe Ile Il - #e Tyr Arg Asp Thr Pro              595          - #       600          - #       605                      - - Val Val Lys Ser Ser Ser Arg Glu Leu Cys Ty - #r Ile Ile Leu Ala Gly          610              - #   615              - #   620                          - - Ile Cys Leu Gly Tyr Leu Cys Thr Phe Cys Le - #u Ile Ala Lys Pro Lys      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Gln Ile Tyr Cys Tyr Leu Gln Arg Ile Gly Il - #e Gly Leu Ser Pro        Ala                                                                                             645  - #               650  - #               655             - - Met Ser Tyr Ser Ala Leu Val Thr Lys Thr As - #n Arg Ile Ala Arg Ile                  660      - #           665      - #           670                  - - Leu Ala Gly Ser Lys Lys Lys Ile Cys Thr Pr - #o Lys Pro Arg Phe Met              675          - #       680          - #       685                      - - Ser Ala Cys Ala Gln Leu Val Ile Ala Phe Il - #e Leu Ile Cys Ile Gln          690              - #   695              - #   700                          - - Leu Gly Ile Ile Val Ala Leu Phe Ile Met Gl - #u Pro Pro Asp Ile Met      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - His Asp Tyr Pro Ser Ile Arg Glu Val Tyr Le - #u Ile Cys Asn Thr        Thr                                                                                             725  - #               730  - #               735             - - Asn Leu Gly Val Val Thr Pro Leu Gly Asn As - #n Gly Leu Leu Ile Leu                  740      - #           745      - #           750                  - - Ser Cys Thr Phe Tyr Ala Phe Lys Thr Arg As - #n Val Pro Ala Asn Phe              755          - #       760          - #       765                      - - Pro Glu Ala Lys Tyr Ile Ala Phe Thr Met Ty - #r Thr Thr Cys Ile Ile          770              - #   775              - #   780                          - - Trp Leu Ala Phe Val Pro Ile Tyr Phe Gly Se - #r Asn Tyr Lys Ile Ile      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Thr Met Cys Phe Ser Val Ser Leu Ser Ala Th - #r Val Ala Leu Gly        Cys                                                                                             805  - #               810  - #               815             - - Met Phe Val Pro Thr Val Tyr Ile Ile Leu Al - #a Lys Pro Glu Arg Asn                  820      - #           825      - #           830                  - - Val Arg Ser Ala Phe Thr Thr Ser Thr Val Va - #l Arg Met His Val Gly              835          - #       840          - #       845                      - - Asp Gly Lys Ser Ser Ser Ala Ala Ser Arg Se - #r Ser Ser Leu Val Asn          850              - #   855              - #   860                          - - Leu Trp Lys Arg Arg Gly Ser Ser Gly Glu Th - #r Leu Arg                  865                 8 - #70                 8 - #75                            - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 343 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: both                                                        (D) TOPOLOGY: both                                                   - -     (ii) MOLECULE TYPE: cDNA                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..343                                                          (D) OTHER INFORMATION: - #/note= "Partial sequence of MGLUR2                       - 3' - #untranslated sequence."                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - TGGAGACGCC ATACTGCCGC GCTGACACAG CTGCTCCTGG GCACCTAGTG CA -             #GACCCACG     60                                                                 - - TCCAGGGCCA GGAGGAAGTT GGCTGGAGCA CTGCAATAAT TTATTACCCA GC -            #CTATGTCT    120                                                                 - - GCCCCCCGAG TCACTTACCC ACCTCCTTAC CCCAGCTCTT CAGACTCAGA AG -            #TCAGGAGC    180                                                                 - - CTTGGCCAGG AGCCTCTGCA GTGGCCACTA ACTGCCCTTG TAGCTGTGTT TC -            #CTCCTGGC    240                                                                 - - CAGGCCCAGG GCTCAGAGAG GAGCAAGCCA GGGTTCACTC TGCCCTGGAC CC -            #GGGTGGCT    300                                                                 - - GAGGACGGCA GGCCCCAGTC CTAACCAGCA AAGGTGCTTC CAG    - #                      - #343                                                                   __________________________________________________________________________

That which is claimed is:
 1. A cation-based bioassay for monitoringreceptor-induced changes in intracellular cyclic nucleotide levels, saidbioassay comprising:introducing a polynucleic acid encoding a humanmetabotropic glutamate receptor into mammalian or Xenopus host cells,that contain at least one type of endogenous or recombinant cyclicnucleotide-gated channel, thereby producing modified host cells whereinsaid receptor is encoded by a polynucleotide that hybridizes under highstringency conditions to a nucleic acid encoding the amino acid sequenceset forth in SEQ ID NO. 4 or SEQ ID NO. 6; and said receptor isexpressed by said modified host cells; and measuring changes in theamount of cyclic nucleotide activation of said cyclic nucleotide-gatedchannels in the presence and absence of a potential ligand for saidreceptor.
 2. bioassay of claim 1 wherein said receptor is encoded by apolynucleotide that hybridizes under high stringency conditions to anucleic acid encoding the amino acid sequence set forth in SEQ ID NO. 4.3. bioassay of claim 1 wherein said receptor is encoded by apolynucleotide that hybridizes under high stringency conditions to anucleic acid encoding the amino acid sequence set forth in SEQ ID NO. 6.4. The bioassay of claim 1 wherein said host cells are selected from thegroup consisting of COS cells, mouse L cells, Chinese hamster ovarycells, human embryonic kidney cells, African green monkey cells, Ltk-cells, BHK cells, and Xenopus laevis oocytes.
 5. The bioassay of claim 1wherein said polynucleic acid further comprises a promoter operativelylinked to said polynucleic acid encoding said receptor, wherein saidpromoter is selected from the group consisting of SV40 promoters,cytomegalovirus promoters, mouse mammary tumor virus promoters, Roussarcoma virus promoters, T7 bacteriophage promoters, and Moloney murineleukemia virus promoters.
 6. The bioassay of claim 1 wherein saidmeasuring step comprises electrophysiological measurement of ion fluxcurrents, fluorescence measurement of intracellular calcium, or singlecell video imaging.
 7. The bioassay of claim 1 wherein said potentialligand is selected from the group consisting of quisqualate, glutamate,trans-1-amino-cyclopentane-1,3-dicarboxylic acid,1S,3R-1-amino-cyclopentane-1,3-dicarboxylic acid,2-amino-3-phosphonopropionate, 2-amino-5-phosphonopentanoate, and6-cyano-7-nitroquinoxaline-2,3-dione.
 8. The bioassay of claim 1 furthercomprising:providing a second host cell that contains said cyclicnucleotide-gated channel, with the proviso that said second host celldoes not express said receptor; measuring changes in the amount ofcyclic nucleotide activation of said cyclic nucleotide-gated channels insaid second host cell in the presence and absence of said potentialligand; and comparing said changes in the amount of cyclic nucleotideactivation of said cyclic nucleotide-gated channels in said second hostcell with the amount of cyclic nucleotide activation of said cyclicnucleotide-gated channels in said modified host cell.
 9. A cation-basedbioassay for monitoring receptor-induced changes in intracellular cyclicnucleotide levels, said bioassay comprising:introducing a polynucleicacid encoding a human metabotropic glutamate receptor into mammalian orXenopus host cells, that contain at least one type of endogenous orrecombinant cyclic nucleotide-gated channel, thereby producing modifiedhost cells, wherein said polynucleic acid hybridizes under highstringency conditions to the nucleic acid sequence set forth in SEQ IDNO. 3 or SEQ ID NO. 5; and said receptor is expressed by said modifiedhost cells; and measuring changes in the amount of cyclic nucleotideactivation of said cyclic nucleotide-gated channels in the presence andabsence of a potential ligand for said receptor.
 10. The bioassay ofclaim 9 wherein said polynucleic acid hybridizes under high stringencyconditions to the nucleic acid sequence set forth in SEQ ID NO:3. 11.The bioassay of claim 9 wherein said polynucleic acid hybridizes underhigh stringency conditions to the nucleic acid sequence set forth in SEQID NO:5.
 12. The bioassay of claim 9 wherein said host cells areselected from the group consisting of COS cells, mouse L cells, Chinesehamster ovary cells, human embryonic kidney cells, African green monkeycells, Ltk- cells, BHK cells, and Xenopus laevis oocytes.
 13. Thebioassay of claim 9 wherein said polynucleic acid further comprises apromoter operatively linked to said polynucleic acid encoding saidreceptor, wherein said promoter is selected from the group consisting ofSV40 promoters, cytomegalovirus promoters, mouse mammary tumor viruspromoters, Rous sarcoma virus promoters, T7 bacteriophage promoters, andMoloney murine leukemia virus promoters.
 14. The bioassay of claim 9wherein said measuring step comprises electrophysiological measurementof ion flux currents, fluorescence measurement of intracellular calcium,or single cell video imaging.
 15. The bioassay of claim 9 furthercomprising:providing a second host cell that contains said cyclicnucleotide-gated channel, with the proviso that said second host celldoes not express said receptor; measuring changes in the amount ofcyclic nucleotide activation of said cyclic nucleotide-gated channels insaid second host cell in the presence and absence of said potentialligand; and comparing said changes in the amount of cyclic nucleotideactivation of said cyclic nucleotide-gated channels in said second hostcell with the amount of cyclic nucleotide activation of said cyclicnucleotide-gated channels in said modified host cell.